Biodegradable tissue composition with biodegradable cross-linkers

ABSTRACT

Novel implantable tissue fixation methods and compositions are disclosed. Methods and compositions of tissue, fixed using polymeric and/or variable length crosslinks, and di- or polymercapto compounds are described. Also described are the methods and compositions wherein the tissue is fixed using biodegradable crosslinkers. Methods and compositions for making radio-opaque tissue are also described. Methods and compositions to obtain a degradable implantable tissue-synthetic biodegradable polymer composite are also described. Compositions and methods of incorporating substantially water-insoluble bioactive compounds in the implantable tissue are also disclosed. The use of membrane-like implantable tissue to make an implantable drug delivery patch are also disclosed. Also described are the compositions and methods to obtain a coated implantable tissue. Medical applications implantable tissue such as heart valve bioprosthesis, vascular grafts, meniscus implant, drug delivery patch are also disclosed.

This application is a continuation of U.S. patent application Ser. No.13/075,039 filed Mar. 29, 2011, which is a continuation of U.S. patentapplication Ser. No. 11/661,062 filed Feb. 22, 2001 which is a nationalstage entry of PCT Application No. PCT/US2005/030187 filed Aug. 24,2005, which claims benefit of priority to U.S. Provisional ApplicationNo. 60/604,737, filed Aug. 26, 2004, each of which are herebyincorporated by reference herein in their entirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for thepreparation of biological tissues or extracellular matrices and theirmedical applications.

BACKGROUND OF THE INVENTION

The use of human or animal tissue for medical or surgical use is arapidly growing therapeutic field. Many uses of processed biologicaltissues for implantation into humans have been reported. The commercialproducts or products under development include wound healing dressings,tissue heart valves, ligament substitutes, pericardial patches andmembranes, vascular grafts and the like. The use of animal tissue offersan inexpensive source of materials to fabricate tissue-based medicalproducts. The problems with the animal tissue transplantation includeinflammation, unwanted degradation, control over the degradationprocess, calcification, inability to release bioactive compounds in acontrolled manner, and rejection of the transplanted tissue.

The primary component of many biological tissues is a protein calledcollagen. Collagen-based biomaterials generally induce a mildinflammatory response, which results in degradation of the protein. Thisdegradation can be prevented by chemical modification or crosslinking oftissue proteins and is achieved by reacting difunctional andpolyfunctional crosslinkers capable of forming irreversible and stableintermolecular chemical crosslinking between two collagen chains.Chemical crosslinking may also increase strength and durability of thetissue. Many heart valve bioprosthesis manufacturers use glutaraldehydeas a crosslinking agent for stabilization of the bioprosthesis tissue.The chemistry of glutaraldehyde is complex but well documented.Glutaraldehyde reacts with free amine groups from lysine residues oncollagen and forms Schiff base addition products. Althoughglutaraldehyde is the most commonly used chemical fixative forbiological tissues, there are a number of drawbacks associated with itsuse in the production of bioprosthetic devices. For example, the longterm durability of glutaraldehyde-fixed bioprostheses is not wellestablished. Another drawback to glutaraldehyde fixation ofbioprostheses relates to the release of cytotoxic glutaraldehyde on thetissue surface thereby hindering the growth of cells, especiallyendothelial cells, on the surface of the tissue. Glutaraldehyde fixedtissue is also susceptible to calcification which leads to devicefailure.

To overcome limitations of glutaraldehyde crosslinking, other chemicalcrosslinking agents capable of reacting with amine, carboxyl andhydroxyl group have been explored. Tissue crosslinking chemistry hasbeen recently reviewed. However, none of alternative chemistries haveresulted into a commercial clinical heart valve product. Tissuecrosslinking chemistry is challenging due to a variety of reasons. Fromthe chemistry point of view, the crosslinking reaction is aheterogeneous reaction, where the reactant (tissue) is always in aseparate phase (solid state phase) as compared to the crosslinker(solution, oiled or liquid phase). This limits the accessibility oftissue functional groups for crosslinking reaction. The solid statenature of tissue also makes it difficult for large crosslinker moleculessuch as, by way of example, and not limitation, polymers to penetrateinside the tissue matrix and crosslink the reactive sites. Generally,the crosslinking reaction must be done without denaturing the protein.The denatured tissue/collagen (gelatin) is more susceptible to enzymaticdegradation and denatured proteins have inferior mechanical propertiesas compared to non-denatured tissue. To prevent denaturing of tissue,the use of aggressive organic solvents and high temperatures in tissuecrosslinking is generally avoided. It is generally believed that anaqueous medium with physiological conditions (pH 7.2, 37° C.) is bestsuited for tissue crosslinking. Fixation under physiological conditionsis most likely to preserve the natural conformation of proteins presentin the tissue. Glutaraldehyde is one of the few crosslinking agentscapable of reacting with the tissue in water under physiologicalconditions.

In known approaches, most of the tissue crosslinking is restricted todi- or polyfunctional small compounds such as, by way of example, andnot limitation, glutaraldehyde. Small compounds can easily penetratesolid tissue matrix and can crosslink surface as well as bulk componentsof the tissue matrix. In order for crosslinking to occur, two or morereactive functional groups must react with two polymeric chains to forman interchain crosslinked moiety. Most tissue crosslinkers are singlechemical entities and therefore have fixed molecular length. The fixedlength crosslinker can only react with those sites which are within theclose proximity of its reactive functional groups. Therefore, it cannotcrosslink the tissue if the reactive sites present on the tissue are ata shorter or longer distance than the length of crosslinker. Also,during the crosslinking reaction, one of the crosslinking functionalgroup reacts with the crosslinkable moiety such as, by way of example,and not limitation, collagen. After the reaction, the other functionalgroup must react with other reactive site on the collagen to completethe crosslinking reaction. This often may not be possible due to limitedlength and mobility of crosslinker. This results in a number of danglingbonds with incomplete crosslinking. Therefore, the length of acrosslinker serves as a major limitation in achieving a high degree ofcrosslinking. Thus, there is a need for tissue crosslinking methodswherein the crosslinks formed may have variable lengths.

Polymeric crosslinkers can be useful in crosslinking the tissue due tohigh molecular flexibility of polymeric molecular coil and polymer'sability to impart additional properties to the tissue matrix. However,polymeric crosslinkers are large molecules which cannotdiffuse/penetrate inside the tissue matrix and react with sites presentin the bulk of the tissue. This limits the ability of polymericcrosslinker to surface crosslinking only. Known techniques generally donot teach the successful use of polymeric crosslinkers in tissuecrosslinking. There is a need for methods and compositions that permitthe incorporation and crosslinking of tissue using polymers or thatgenerate polymeric crosslinks.

Shape memory biomaterials have the ability to change to a predeterminedshape when subjected to an appropriate energy stimulus. Nitinol alloy isone of the well-known shape memory biomaterials. Many applications ofNitinol materials have been commercialized. These applications includeperipheral vascular stent and stent grafts, vena cava filters, etc.Bioprosthetic tissues having shape memory properties can be extremelyuseful in making novel medical devices. There is a need for tissue-basedbiomaterials that can remember shape maintained during fixation orstabilization and tissue-based materials with the ability to rememberand recover the shape when deformed by a mechanical force.

Unfixed or non-crosslinked animal tissue undergoes enzymatic degradationwhen implanted in human/animal body. Usually such degradation isfollowed after a moderate to severe inflammatory response; presumablydue to an immunological reaction to the foreign biological materials inthe host body. Non-crosslinked animal tissue such as, by way of example,and not limitation, porcine small-intestinal submucosa has beencommercialized as a wound dressing material. In many medicalapplications, it is desirable to have a biological degradable tissuewith no or little inflammatory response and control over its degradationprofile and properties. Known techniques generally do not teach methodsand compositions that will affect the degradation behavior of biologicaltissue. Therefore, there is also a need for methods and compositionsthat can reduce the inflammatory response to the animal or human tissue.Compositions and methods that will control the degradation time of theimplanted tissue are also needed.

Animal tissue used in commercial bioprostheses such as heart valve,vascular graft and vascular patch is limited by tissue thickness, sizeand protein (chemical) composition. For example, bovine pericardium, awidely used animal tissue has a thickness ranging 1 to 2 mm which maytoo thick for some medical applications such as low profile stent graftapplication. The useful tissue recovered from one animal is also limitedin size. Typical area of bovine pericardial or porcine pericardialtissue may range from 50 to 150 square inches. This size and thicknesslimitation may limit the use of tissue in making large medical devicesuch as tissue based dialysis catheters. The tissue size limits alsoincreases production costs due to lower yields. The higher size ofimplantable tissue may permit to manufacture more devices per tissue andreduce manufacturing costs. Therefore there is need for tissue,especially membrane like tissue, which can be made in wide ranges ofsize, thickness and with different chemical compositions forbioprosthesis applications.

Synthetic biodegradable polymers have received considerable interest inthe medical and pharmaceutical field at least because they can performtemporary therapeutic functions and are eliminated from the body oncetheir therapeutic function has been accomplished. Some of the well-knownapplications of biodegradable polymers include surgical sutures,staples, or other wound closure devices, as a carrier for bioactivesubstances for controlled drug delivery, etc. Several types ofbiodegradable polymers have been reported in the subject literature,however, polymers prepared from hydroxy acids have received muchattention due to their degradability and toxicological safety.Homopolymers and copolymers based on the l-lactic acid, dl-lactic acidand glycolic acid are among the most widely used polymers for medicalapplications. These polymers can be formulated into variety of physicalforms such as, by way of example, and not limitation, fibers orfilaments with acceptable mechanical properties and degradation profileand nontoxic degradation products. Synthetic biodegradable polymers suchas, by way of example, and not limitation, polyanhydrides, polylactones,and polyhydroxyacids have been extensively investigated for controlleddrug delivery applications as well as for a scaffold for tissueengineering. These polymers can release a bioactive compound uponbioerosion and thus permit localized controlled therapeutic delivery.There is a need for biological tissue, preferably degradable biologicaltissue, which can release a bioactive compound in a controlled manner,preferably using a hydrolysis or bioerosion mechanism. There is also aneed for materials which can provide properties of syntheticbiodegradable material and biological tissues.

Polyethylene oxide (PEO) or polyethylene glycol (PEG) is a water solublebiocompatible polymer which is being used in several commerciallyavailable pharmaceutical and medical products. PEG is water soluble andnon-ionic in nature. When injected in human or animal body, it israpidly cleared by the body. When it is immobilized either physically orchemically on a polymer surface, it renders the surface highly resistantto protein adsorption. The resistance to protein adsorption is believedto be responsible for reduced bacterial and cell adhesion to PEG-richsurfaces. The reduction in protein adsorption also increases thebiocompatibility of blood- and tissue-contacting materials. Hydrated PEGchain is not recognized by the immune system, therefore it is used toreduce the immunogenicity and antigenicity of proteins and henceincrease their circulation time. Nonionic hydrogels, such as, by way ofexample, and not limitation, the poly(ethylene glycol) (PEG)-basedhydrogels, are biocompatible and are non-cell adhesive. Tissue-basedbioprostheses which combine the properties of PEG and biological tissuemay be useful for many medical applications.

In view of the foregoing, there is a need for compositions and methodsthat provide biostable implantable tissue. There is also a need forbiodegradable biological tissue with control over its degradation timeand with the ability to release bioactive compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is illustrated by way of example, and not by wayof limitation, in the figures of the accompanying drawings and in whichlike reference numerals refer to similar elements and in which:

FIG. 1 is a schematic representation of exemplary steps involved intissue crosslinking by free radical polymerization, in accordance withan embodiment of the present invention;

FIG. 2 is an exemplary reaction scheme for tissue crosslinking usingacrylic acid n-hydroxysuccinimide (NHS) ester, in accordance with anembodiment of the present invention;

FIG. 3 is an exemplary reaction scheme for tissue crosslinking using di-or polyunsaturated acid n-hydroxysuccinimide (NHS) ester, in accordancewith an embodiment of the present invention;

FIG. 4 is an exemplary reaction scheme for tissue crosslinking usingunsaturated aldehydes, in accordance with an embodiment of the presentinvention;

FIG. 5 is a schematic representation of the exemplary preparation oftissue with patterns of biostable and biodegradable regions within thetissue, in accordance with an embodiment of the present invention;

FIG. 6 is a schematic representation of an exemplary shape-preservingtissue fixation method, in accordance with an embodiment of the presentinvention.

FIG. 7 is a schematic representation of exemplary steps involved intissue crosslinking using mercapto compounds, in accordance with anembodiment of the present invention;

FIG. 8 is a schematic representation of an exemplary tissue crosslinkingusing biodegradable tissue crosslinker, in accordance with an embodimentof the present invention;

FIG. 9 is a schematic representation of an exemplary biodegradableuncrosslinked tissue modified using polyethylene glycol, in accordancewith an embodiment of the present invention;

FIG. 10 is an exemplary reaction scheme for tissue or collagenmodification with polyhydroxyacid or polylactones, in accordance with anembodiment of the present invention.

FIG. 11 is a schematic representation of an exemplary membrane-liketissue modifications (A-F), in accordance with an embodiment of thepresent invention;

Unless otherwise indicated illustrations in the figures are notnecessarily drawn to scale.

SUMMARY OF THE INVENTION

To achieve the forgoing and other objects and in accordance with thepurpose of the invention, a variety of implantable tissue compositionsand methods thereof are described.

One embodiment of the present invention provides a composition ofmatter, comprising an uncrosslinked biological tissue, wherein thetissue is chemically modified with unsaturated polymerizable groups

Another embodiment of the present invention provides a composition ofmatter comprising a biological tissue modified with unsaturated groups,wherein unsaturated groups are used in chemical crosslinking of thetissue.

Another embodiment of the present invention provides a composition ofmatter comprising a biological tissue modified with unsaturated groups,wherein the cross-linked biological tissue is produced by treating thetissue under effective cross-linking condition comprising a free radicalinitiator or photoinitiator. Preferably the crosslinking is done inpresence of a mono or polyunsaturated compound capable of copolymerizingwith the unsaturated groups in the tissue.

Another embodiment of the present invention provides a composition ofmatter comprising a biological tissue modified with unsaturated groups,wherein unsaturated groups are copolymerized with free radicalpolymerizable comonomers. The comonomers may include functional monomerswith reactive functional groups such as epoxide or isocyanate; monomerswith charged groups; monomers that undergo crosslinking andbiodegradation; monomers that produce thermosensitive polymers; monomerswith long alkyl chains; monomers that produce crystalline orsernicrystalline polymers; monomers that produce functional polymersupon hydrolysis such as polyvinyl alcohol and monomers that haveradio-opaque moieties.

Another embodiment of the present invention provides a composition ofmatter comprising a biological tissue modified and crosslinked withunsaturated groups, wherein unsaturated modified groups and/orcrosslinks with unsaturated groups are copolymerized with free radicalpolymerizable comonomers.

Another embodiment of the present invention provides a composition ofmatter comprising a biological tissue modified with unsaturated groups,wherein unsaturated groups are copolymerized with free radicalpolymerizable comonomers that have biodegradable or hydrolizable groups.The biodegradable monomers may be hydrophilic or hydrophobic.

Another embodiment of the present invention provides a composition ofmatter comprising a biological tissue modified with unsaturated groups,wherein a cross-linked biological tissue is produced by treating theunsaturated groups modified tissue under effective cross-linkingconditions with an organic di or poly-mercapto compounds. Preferably,the di or poly-mercapto organic compound is a solute in a fluidcomprising a solvent.

Another embodiment of the present invention provides a composition ofmatter comprising a membrane like biological tissue and a surgicaladhesive. The membrane like tissue and surgical adhesive are formulatedto form a “surgical adhesive patch”. The surgical adhesive patch couldbe biodegradable.

Yet another embodiment of the present invention relates to across-linked biological tissue produced by treating the tissue undereffective cross-linking conditions with a biodegradable crosslinker.Preferably, the biodegradable crosslinker is a solute in a fluidcomprising a solvent.

Yet another embodiment of the present invention provides a compositioncomprising a radio-opaque implantable animal tissue.

Yet another embodiment of the present invention relates to a biologicaltissue having shape memory properties.

Yet another embodiment of the present invention relates to a biologicaltissue wherein certain parts or regions of the tissue are made biostablewhile the remaining parts of the tissue are made biodegradable. Thebiostable and biodegradable regions with in the tissue can be of anygeometry.

Yet another embodiment of the present invention relates to anon-crosslinked degradable biological tissue produced by treating thetissue under effective treatment conditions with a monofunctional regentcapable of reacting with primary amine groups on the tissue. Preferably,the monofunctional reagent is: a polyether derivative or an activatedacid derivative such as n-hydroxysuccinimide derivative, or a cycliclactide such as glycolide or lactide or an isocyanate derivative or ananhydride derivative.

Yet another embodiment of the present invention relates to thesubstantially biostable or biodegradable tissue produced by treating thetissue under effective treatment conditions with a cyclic lactone toproduce a tissue-polylactone graft copolymer.

Yet another embodiment of the present invention relates to a biologicaltissue/synthetic biodegradable polymer composite produced by treatingthe tissue with a fluid comprising synthetic biodegradable polymer.

Yet another embodiment of the present invention relates to a biologicaltissue synthetic biodegradable polymer composite wherein syntheticbiodegradable polymer is chemically bonded to the biological tissue.

Another embodiment of the present invention relates to a degradablebiological tissue-synthetic biodegradable polymer composite produced bytreating a non-crosslinked tissue under effective treatment conditionswith a synthetic biodegradable polymer. Preferably, the syntheticbiodegradable polymer is polylactone or polyhydroxyacid derivative in afluid comprising a solvent. The synthetic biodegradable polymer can alsobe a crosslinked polymer.

Yet another embodiment of the present invention relates to a biologicaltissue produced by treating the tissue with a fluid comprising syntheticbiodegradable polymer and a bioactive compound.

Yet another embodiment of the present invention provides a compositionof matter that promotes the localized controlled delivery of at leastone drug.

Yet another embodiment of the present invention provides an animaltissue based controlled drug delivery patch that releases at least onebioactive compound.

Yet another embodiment of the present invention relates to a method ofcross-linking a tissue is provided. The method comprises: linking thefree radical polymerizable groups on the tissue with a covalent bond;crosslinking the free radical polymerizable groups using free radicalmechanism or cyclic dimerization.

Yet another embodiment of the present invention relates to a method ofcross-linking a tissue is provided. The method comprises: covalentlylinking the compounds containing at least one free radical polymerizablegroup on the tissue; crosslinking the free radical polymerizable groupusing a di or poly-mercapto organic compounds.

Yet another embodiment of the present invention relates to a method ofcross-linking a tissue is provided. The method comprises: crosslinkingthe tissue with compounds containing at least one free radicalpolymerizable group; further crosslinking the free radical polymerizablegroup using free radical chemistry such as free radical dimerization andpolymerization, free radical crosslinking or free radicalcopolymerization with monomer.

In still another embodiment of the present invention, a method formaking a biodegradable biological tissue is provided. The methodcomprises: treating the tissue under effective cross-linking conditionswith a fluid comprising a biodegradable crosslinker.

In still another embodiment of the present invention, a method forincorporating a biodegradable polymer in a biological tissue isprovided. The method comprises: dehydrating the biological tissue;treating the dehydrated tissue with a solution of biodegradable polymerin an organic solvent; removing the solvent from the treated tissue.

In still another embodiment of the present invention, a method forincorporating a biodegradable polymer and a bioactive compound in abiological tissue is provided. The method comprises: dehydrating thebiological tissue; treating dehydrated tissue with a solution ofbiodegradable polymer and bioactive compound in an organic solvent;removing the solvent from the treated tissue.

In still another embodiment of the present invention, a method formaking a drug delivery patch from a membrane like tissue is provided.The method comprises: dehydrating the membrane like biological tissue;treating dehydrated membrane like tissue with a solution ofbiodegradable polymer and a bioactive compound in an organic solvent;removing the solvent from the treated tissue; releasing the compoundfrom the biodegradable polymer. Preferably the bioactive compound is acell cycle inhibitor such as Lovastatin (HMG-CoA inhibitor or statin),paclitaxel, and Rapamycin. The biodegradable polymer may be hydrophobicor hydrophilic. The biodegradable polymer can be a crosslinked polymer.

In still another embodiment of the present invention, a method ofcoating a biological tissue with biodegradable polymer is provided. Themethod comprises: dehydrating the biological tissue; spraying a coatingsolution comprising biodegradable polymer in a solvent; removing thesolvent from the treated tissue.

In still another embodiment of the present invention, a method ofcoating a biological tissue with biodegradable polymer is provided. Themethod comprises: dehydrating the biological tissue; dipping thedehydrated tissue in a coating solution comprising biodegradable polymerin a solvent; removing the solvent from the treated tissue.

In still another embodiment of the present invention, a method formaking a radio-opaque implantable tissue is provided. The methodcomprises: treating a biological tissue with a radio-opaque compoundunder effective treatment conditions to covalently bond the radio-opaquecompound to the tissue. The preferred radio-opaque compound is iodinatedorganic compound.

In still another embodiment of the present invention, a method treatingthe tissue under effective cross-linking conditions with a di orpolymercapto organic compound is provided.

In still another embodiment of the present invention, a method ofcoating a biological implantable tissue with a biodegradable hydrogel isprovided. The method comprises: treating a tissue with a precursor orbiodegradable hydrogel components; crosslinking the precursors toproduce a biodegradable hydrogel coating on the surface of the tissue.

In still another embodiment of the present invention, a method ofcoating a biological implantable tissue with biodegradable hydrogelcomprising cells/bioactive compound is provided. The method comprises:treating a tissue with a precursor or biodegradable hydrogel componentscomprising cells and/or bioactive compounds; crosslinking the precursorsto produce a biodegradable hydrogel coating with entrapped cells/drug inthe coating on the surface of the tissue.

In still another embodiment of the present invention, a method ofcoating a biological tissue with non-crosslinked biodegradable hydrogelis provided. The method comprises: dehydrating the biological tissue;treating the dehydrated tissue with a solution of biodegradable polymerin an organic solvent; removing the solvent from the treated tissue;exposing the tissue to a biological environment to hydrate the tissueand biodegradable polymer.

In still another embodiment of the present invention, a method forincorporating a biodegradable polymer and bioactive substance inbiological tissue is provided. The method comprises; forming groves orholes on tissue surface; filling the grooves or holes with abiodegradable polymer and bioactive compound; releasing the bioactivecompound in a controlled manner.

Another embodiment of the present invention provides a degradable animaltissue coated with or incorporated with, Demineralized Bone Matrix (DBM)and/or purified Bone Marrow Proteins (BMP)'s. This mixture provides amatrix that allows the cellular components of the body to migrate intoit and thus produce osteoinduction where needed. The matrix composition,enzymes (such as thrombin and plasmin), BMPs, growth factors and DBM andtheir concentrations, calcium salts such as calcium phosphates areadequately formulated to optimize the longevity of this temporalscaffolding structure and the osteoinduction which needs to occur. Allof the animal tissue components are biodegradable, but duringosteogenesis the mixture provides a non-collapsible scaffold that candetermine the shape and location of the newly formed bone.

Another embodiment of the present invention provides a composition ofmatter comprising a degradable tissue coated with a biodegradablepolymer comprising at least one growth factor and/or a drug.

Yet another embodiment of the present invention provides a compositionof matter that promotes wound healing, comprising: a biodegradableimplantable animal tissue coated with biodegradable polymer and aneffective concentration of at least one growth factor, wherein theconcentration of growth factor is effective in promoting wound healing.

Another embodiment of the present invention provides a composition ofmatter that promotes the growth of cells, comprising: a degradableanimal tissue; a hydrogel coating on the surface of degradable tissue;and an effective concentration of at least one growth factor, whereinthe concentration of the growth factor is effective in promoting thedirected migration of the animal cells. In another embodiment,genetically altered cells and/or other cells may also be included in thetissue coated hydrogels of this invention.

Yet another embodiment of the present invention provides a compositionof matter that promotes the proliferation and/or differentiation ofanimal cells, comprising: an implantable animal tissue, a hydrogel; andan effective concentration of at least one growth factor, wherein theconcentration is effective in promoting proliferation and/ordifferentiation of animal cells.

Yet another embodiment of the present invention provides a compositionof matter that promotes the localized delivery of at least one growthfactor. Preferably the growth factor is vascular endothelial growthfactor (VEGF) or BMP or mixtures thereof.

Yet another embodiment of the present invention provides a process forpromoting the healing of wounds, comprising applying to the wound, acomposition that contains a non-crosslinked animal degradable animaltissue modified with a synthetic polymer and an effective concentrationof at least one growth factor or one small molecule therapeutic, whereinthe concentration is effective to promote wound healing.

Another embodiment of the present invention provides a degradableimplantable animal tissue based composition that promotes the localizeddelivery of a poorly water soluble form of a bioactive compound, such aschlorhexidene; chlorhexidene diacetate monohydrate or chlorhexidenedihydrochloride; chlorhexidene gluconate, silver salts such as silverchloride, silver iodide, silver acetate, silver lactate, cell cycleinhibitor such as paclitaxel, lovastatin, rapamycin, simvastatin,rifampin; or anti-arrhythmic agent such as amiodarone.

In still another embodiment of the present invention, a method fortissue crosslinking or fixation is provided. The method comprises;linking the free radical polymerizable groups on the tissue with acovalent bond; crosslinking the free radical polymerizable groups usingfree radical polymerizable monomers comprising primary amine group.Further crosslinking the primary amine groups using di or polyfunctionalcrosslinker such as glutaraldehyde.

In yet another embodiment of the present invention, method for making adegradable tissue matrix comprising substantially water insoluble drugor bioactive compound is provided. The method comprises: dehydrating themembrane like biological tissue; treating dehydrated membrane liketissue with a solution of a substantially water insoluble bioactivecompound in an organic solvent; removing the solvent from the treatedtissue.

In still another embodiment of the present invention, a method formaking a tissue capable of remembering the shape is provided.

In another embodiment, a In another embodiment of the present invention,a method for making a tissue is provided wherein certain parties of thetissue are biostable and/or biodegradable.

Other features, advantages, and object of the present invention willbecome more apparent and be more readily understood from the followingdetailed description, which should be read in conjunction with theaccompanying drawings.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is best understood by reference to the detailedfigures and description set forth herein.

Embodiments of the invention are discussed below with reference to theFigures. However, those skilled in the art will readily appreciate thatthe detailed description given herein with respect to these figures isfor explanatory purposes as the invention extends beyond these limitedembodiments.

In order to clarify the terminology present invention, the followingdefinitions are provided. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as is commonlyunderstood by one who is skilled in the art.

“Biodegradable” denotes a material that will degrade in a biologicalenvironment by either a biologically assisted mechanism, such as, by wayof example, and not limitation, an enzyme catalyzed reaction, or by achemical mechanism which can occur in a biological medium, such as, byway of example, and not limitation, hydrolysis.

“Biostable” denotes a high chemical stability of a compound in anaqueous environment, which is similar to the environment, found in thehuman body, such as, by way of example, and not limitation, phosphatebuffered saline (pH 7.2).

“Bioactive” refers to one or all of the activities of a compound thatshow pharmacological or biological activity in human or animal body.Such biological activity is preferred to have therapeutic effect.Substances or compounds that are bioactive are referred to as “drugs” or“bioactive compounds.” The bioactive compounds that can be used include,but are not limited to, antiviral agents; antiinfectives such as, by wayof example, and not limitation, antibiotics; antipruritics;antipsychotics; cholesterol- or lipid-reducing agents; cell cycleinhibitors; anticancer agents; antiparkinsonism drugs; HMG-CoAinhibitors; antirestenosis agents; antiinflammatory agents;antiasthmatic agents; anthelmintic; immunosuppressives; musclerelaxants; antidiuretic agents; vasodilators; nitric oxide; nitricoxide-releasing compounds; beta-blockers; hormones; antidepressants;decongestants; calcium channel blockers; growth factors such as, by wayof example, and not limitation, bone growth factors or bone morphogenicproteins; wound healing agents; analgesics and analgesic combinations;local anesthetic agents; antihistamines; sedatives;angiogenesis-promoting agents; angiogenesis-inhibiting agents;tranquilizers and the like. Cellular elements which can be used fortherapeutic use include, but are not limited to mammalian cellsincluding stem cells, cellular components or fragments, enzymes, DNA,RNA, and genes may also be included as bioactive components.

The term “minimally invasive surgery” or (MIS) is used herein includes,but is not limited to, surgical techniques such as, by way of example,and not limitation, laparoscopy, thoracoscopy, arthroscopy, intraluminalendoscopy, endovascular techniques, catheter-based cardiac techniques(such as, by way of example, and not limitation, balloon angioplasty),and interventional radiology.

The term “dehydration” broadly refers to any method that removes thewater from the tissue without denaturing the tissue. This includes, butis not limited to processes such as, by way of example, and notlimitation, lyophilization, vacuum drying, air drying, or solvent-baseddrying such as, by way of example, and not limitation, exposing thetissue to various alcohol-based solutions.

“Sustained release” or “long term release or deliveries” are phrasesused interchangeably herein, to denote longer than the expected deliveryof a bioactive compound from the tissue matrix. In some embodiments,delivery will be at least a day or more, and may extend to weeks,months, or a few years. However, alternate embodiments encompass shorterterm release. The long term release can be achieved by any of a numberof mechanisms.

A “hydrogel” refers to a semisolid composition constituting asubstantial amount of water, and in which polymers or mixtures thereofare dissolved or dispersed. The hydrogels may be physically orchemically crosslinked.

The term “fluid” generally refers to solutions, emulsions, andsuspensions.

The term “exposing” refers to soaking the tissue in a fluid comprisingthe treatment agent for a period of time sufficient to treat the tissue.The soaking may be performed by, but is not limited to, incubation,swirling, immersion, mixing, or vortexing.

Polymeric nomenclature variations such as, by way of example, and notlimitation, poly(lactic acid), polylactic acid, or polylacticacid referto the same polymer, unless or otherwise stated clearly. This is alsotrue for all others polymers referred to herein.

The term “macromonomer or “macromer” refers to oligomeric or polymericmaterials capable of undergoing fee radical polymerization.

The term “activated” means increasing the chemical reactivity of a givenfunctional group so that it can react with the target molecule undermild conditions. By way of example and not limitation, the acidfunctionality in acrylic acid is not reactive enough to react with aminegroups of the tissue in water at pH 7.2 at room temperature. Thereactivity of acid group can be increased sufficiently so that it canreact with proteins or tissue in water at pH 7.2 at room temperature.This may be achieved by forming n-hydroxy succinimide ester of acidfunctional group. Many activation chemistries are known in the peptidesynthesis or protein modification art and will be apparent to oneskilled in the art, in light of the teachings of the present invention.Preferred activating moieties include, but are not limited to,disuccinimidyl moieties, n-hydroxy disuccinimidyl moieties,sulfo-disuccinimidyl moieties, and mixtures thereof.

The phrase “effective cross-linking condition” generally refers to, butis not limited to, exemplary conditions such as treating a biologicaltissue like bovine pericardium tissue (size 2 cm by 2 cm) with 20 ml0.4% glutaraldehyde solution in distilled water or in 20 mM phosphatebuffered saline (PBS, pH 7.2) for 24 to 48 hours at room temperature(around 25° C.).

“In situ” denotes at a local surgical site, especially within or incontact with living organisms, tissue, organs, or the body.

“Biodegradable polymer” denotes those polymers or macromolecules whichdegrade or hydrolyze inside the human or animal body without producingharmful degradation products. Polylacticacid or poly(lactic acid) orpoly(lactide) or PLA is term used for a polymer which is made fromlactide or lactic acid. Similarly, PGA is term used for polyglycolicacid or polyglycolate. Such polymers are generally referred to aspolylactones or polyhydroxyacids.

The term “hydrophobic” is defined as materials or polymers having a lowdegree of water absorption or attraction.

The term “hydrophilic” is defined as materials or polymers having astrong affinity for water.

The term “Tissue Engineering” is defined as the use of a combination ofcells, engineering materials, and suitable biochemical factors tosynthesize biological tissues.

The term “tissue” or “extracellular matrix” (ECM) includes human oranimal tissue suitable for human or animal implantation or medical use.The tissue defined herein includes but is not limited to items such as,by way of example, and not limitation, ligaments; meniscus; basalmembrane; bladder tissue, tendons; cartilage tissue; tubular tissue suchas, by way of example, and not limitation, arterial tissue and veintissue; heart valve tissue; demineralized bone tissue; tissues used toconstruct heart valves such as, by way of example, and not limitation,dura mater and pericardium tissue; transparent issue such as, by way ofexample, and not limitation, cornea and lens tissue; membrane-liketissue such as, by way of example, and not limitation, porcinepericardium; bovine pericardium; porcine intestine tissue and lungtissue, more specifically porcine sub-mucosa tissue; bladder tissue;human tissue that is generated and discarded during human childbirth,e.g., human placenta and umbilical cord tissue generated during childbirth; amniotic membrane tissue; and the like. Among the tubular tissue,tissue such as, by way of example, and not limitation, bovine mesentericvein, bovine thoracic artery, bovine ureter, sheep carotid arteries areused in preferred embodiments. The tissue can be derived from, but isnot limited to, sources such as, by way of example, and not limitation,bovine, human, porcine, horse, sheep, kangaroo, rat, mouse, dog, cat, orrabbit. Tissue derived from human source is generally preferred. Humanprocessed tissue such as AlloDerm® marketed by LifeCell Corporation, NJmay also be used. If the tissue used is from an animal source, theanimal may be genetically modified to yield a uniform tissue type ortissue that has similar biological properties as human tissue. Forexample, the tissue may be obtained from cloned animals. Many cloningtechnologies including those that have already developed or yet to bedeveloped may, for example, and without limitation, be used to obtain acloned animal tissue. The animals may also be genetically modified sothat the antigens which are recognized by the human immune system aresuppressed. The tissue may also be obtained from technologies employedin the tissue engineering art. In one embodiment using a tissueengineering approach, a tissue is generated by growing animal or humanderived cells on a biodegradable scaffold. For example, Apligraf™ is acommercially available human tissue engineering product that may also beused in certain applications. In one embodiment, an engineered tissuethat is provided as a sheet or membrane is used. In some embodiments,tissue that is not denatured may be preferred. Tissue that is acellularor processed to remove cells or cell debris is used in otherembodiments. In some embodiments, it may be preferable to use animaltissue that is substantially free from infectious or harmful animalviruses or proteins such as, by way of example, and not limitation, BSE.

“Bioprosthesis” is defined to include any prosthesis which is derived inwhole or part from animal or other organic tissue and which is suitablefor human or animal implantation. Thus, the term generally includes, butis not limited to, devices such as animal tissue based heart valves,vascular grafts, annuloplasty rings and other medical devices such as,by way of example, and not limitation, vascular grafts or patch, heartreplacements devices, urinary tract and bladder replacements, bowel andtissue resections in general and the like. In general, though notrequired, the type of tissue utilized as the starting material as wellas its modification will depend on the intended use. For example, in theheart valve application, a biostable mechanically durable tissue may,for example, and without limitation, be used. In such an application, abovine pericardium or porcine aortic root that is crosslinked with abiostable crosslinker may, for example, and without limitation, be used.If the tissue is to be used as a biodegradable drug delivery patch ordegradable tissue engineering scaffold, then a non-crosslinked orbiodegradable tissue may, for example, and without limitation, be used.

“Crosslink” is defined as understood by those skilled in thebioprosthesis or polymer chemistry art. In general, cross-linking refersto the method of forming covalent bonds or crosslinks betweenpolymeric/macromolecular molecules. The crosslinking process alsogenerally refers to a fixation process which stabilizes the tissue bymaking the tissue less antigenic and thus less susceptible to enzymaticdegradation. A “crosslinking agent” is defined as a compound capable offorming the crosslinking. For example, glutaraldehyde is generally knownin the art as crosslinking agent for the tissue.

PEG or PEO is a term used for polymer containing ethylene oxide repeatunits.

The term “polymerizable” denotes that molecules have the capacity toform additional covalent bonds resulting in monomer interlinking tooligomer or polymer formation, for example, molecules containcarbon-carbon double bonds of acrylate-type molecules. Suchpolymerization is characteristically initiated by free-radicalformation, for example, resulting from photon absorption of certain dyesand chemical compounds to ultimately produce free-radicals. The termpolymerizable is also applicable to compounds which can undergocondensation polymerization and form a linear or crosslinked polymer.

The following examples are provided as a means of illustrating someembodiments of the present invention and are in no way consideredlimiting of the present invention.

Methods and Compositions for the Preparation of Biostable Tissues.

An exemplary tissue crosslinking method embodiment of the presentinvention is herein provided for biological tissues to be used in, byway of example, and not limitation, bioprosthetic medical devices suchas, by way of example, and not limitation, heart valves, vasculargrafts, surgical patch, ligament and meniscus implants.

One embodiment of the present invention provides for a method ofcrosslinking a tissue comprising steps of treating the tissue undereffective cross-linking conditions with a crosslinker comprisingunsaturated polymerizable groups and further crosslinking theunsaturated polymerizable groups using free radicals. An alternateembodiment of the present invention provides for a method ofcrosslinking a tissue comprising steps of treating the tissue undereffective cross-linking conditions with a crosslinker comprisingunsaturated polymerizable groups and further crosslinking theunsaturated polymerizable groups by free radical copolymerization. Thepresent invention also provides for a method for forming a crosslinkedtissue comprising the steps of providing a tissue suitable for humanimplantation; exposing the tissue to a fluid comprising a functionalmonomer capable of reacting with a tissue until a portion of the tissuefunctional groups are covalently bonded to the monomer; andcopolymerizing the functional monomer-treated tissue under effectivecross-linking conditions with a compound having at least one freeradical polymerizable group. Yet another embodiment of the presentinvention uses a cross-linked biological tissue produced by treating thetissue under effective cross-linking conditions with a biodegradablecrosslinker. In one embodiment, the biodegradable crosslinker may be asolute in a fluid comprising a solvent. It is to be understood that insome applications of the present invention may, for example, and withoutlimitation, be used to produce a general composition of matter and/or abioactive tissue.

As will be set forth in some detail below, one aspect of the presentinvention provides for a composition of matter that promotes thelocalized controlled delivery of at least one drug.

FIG. 1 is a schematic representation of exemplary steps involved intissue crosslinking by free radical polymerization, in accordance withan embodiment of the present invention. Such crosslinking is achieved intwo steps. In the first step, the biological tissue of interest iscontacted with one or more of the disclosed compounds under conditionseffective to introduce unsaturated polymerizable or dimerizable groupsin the tissue, i.e., modified with unsaturated polymerizable groups. Inthe second step, the unsaturated groups are used to form crosslinksbetween components of the tissue, i.e., the unsaturated groups on themodified tissue are copolymerized with free radical polymerizablemonomer and free radical initiator to form a crosslinked tissue with apolymeric crosslink.

The first step shown by way of example in the FIG. 1 for the preparationof tissue with unsaturated groups will next be described in some detail.The unsaturated group modifying agents that may, for example, andwithout limitation, be used in accordance with embodiments of thepresent invention comprise an organic functional small molecule,typically, but not limited to, a monomer with functional groups capableof reacting with tissue. Thus, exemplary compounds suitable for use withthe described embodiments of the present invention can be generallyrepresented by the following structural formula:

F—X—U

Where F is a functional group reactive with proteins present in extracellular matrix such as, by way of example, and not limitation,collagen, elastin, keratin and the like; U is an unsaturated groupcapable of undergoing free radical copolymerization, cyclic dimerizationor reaction with thiol group; and X is an organic molecule or radicalcovalently linking F and U. X may comprise Carbon-Carbon,Carbon-Hydrogen, Carbon-Nitrogen, Carbon-Oxygen, Carbon-Sulfur,Nitrogen-Hydrogen and Oxygen-Hydrogen covalent bonds. X may be polymericor non-polymeric. F is a functional group that is sufficiently reactivewith the components of the tissue such as, by way of example, and notlimitation, collagen or elastin molecules present in the biologicaltissue. For example, functional groups reactive with collagen andsuitable for use may include, but not limited to, anhydride, isocyanate,epoxy, n-hydroxysuccinimide, n-hydroxysulfosuccinimide, aldehyde orother protein reactive functionalities known in the art or yet to bedeveloped. It is preferred to have one F per molecule if the tissuecrosslinking is not desired. If tissue crosslinking is desired, then thenumber of F moieties can be two or more per molecule. If the number of Fmoieties is two or more, the F moieties can be the same functionalgroups (homo-functional) or different functional groups(hetero-functional). U is an unsaturated group capable of undergoingfree radical polymerization or copolymerization. U also can undergo adimerization reaction. U can have at least one carbon-carbon double bondor triple bond capable of undergoing free radical polymerization. Thepreferred unsaturated groups include, but not limited to, acrylamides,methacrylamides, acrylates, diacrylates, oligoacrylates, acrolein,methacrolein, fumarates, maleates, methacrylates, dimethacrylates,oligomethoacrylates, itaconates, cinnamic acid derivatives or otherbiologically acceptable free radical polymerizable groups. The number ofU moieties may be one or more per molecule. The arrangement of X, F andU in the molecule may vary in any order according to various embodimentsof the present invention. There may be more than one F or U group permolecule.

In various embodiments of the present invention, molecules which arecapable of modifying the tissue with unsaturated groups include, but notlimited to, glycidyl methacrylate, glycidyl acrylate, acrylic acidN-hydroxysuccinimide ester, methacrylic acid N-hydroxysuccinimide ester;fumaric acid N-hydroxysuccinimide ester; maleic acidN-hydroxysuccinimide ester; itaconic acid N-hydroxysuccinimide ester;acrylic anhydride, methacrylic anhydride, acryloyl chloride,methacryloyl chloride, 2-isocyanatoethyl methacrylate, 2-hydroxyethylmethacrylate, maleic anhydride, fumaric anhydride, cinnamic acid NHSester, cinnamoyl chloride and the like. Compounds like activatedunsaturated di- or polyfunctional acids such as, by way of example, andnot limitation, fumaric acid N-hydroxysuccinimide ester; maleic acidN-hydroxysuccinimide ester; itaconic acid N-hydroxysuccinimide ester maycrosslink the tissue as well as introduce unsaturated groups. Other dualfunction compounds (i.e., capable of crosslinking and introducingunsaturated groups) will be apparent to one skilled in the art, in lightof the teachings of the present invention.

The tissue modification using functional monomers taught by way ofexample in the present detailed description can be made using anysynthetic methodologies known to the skilled in the art of syntheticpolymer chemistry, in light of the teachings of the present invention.In one embodiment, another aspect of the present invention is directedto a method of introducing an unsaturated group tissue. The methodcomprises treating the tissue under effective treatment conditions witha polymerizable organic compound. In one preferred approach, bovinepericardium tissue is modified with an activated unsaturated acidcompound. The modification method comprises exposing the tissue to afluid comprising activated unsaturated acids that can react with aminogroups in tissue under mild conditions. As used herein, the term“activated” is applied to an acid moiety-containing compound containingan additional moiety such that the activated acid can react with aminogroups under mild conditions. Preferred activating moieties include, butare not limited to, disuccinimidyl moieties, n-hydroxy disuccinimidylmoieties, sulfo-disuccinimidyl moieties, and mixtures thereof. In oneembodiment, bovine pericardium tissue is modified using acrylic acidsuccinimide ester (ANHS). Briefly, bovine pericardium pieces, cut from afreshly obtained bovine pericardial sac, are treated acrylic acidsuccinimide ester (ANHS) dissolved in dimethyl sulfoxide. The reactionis carried out, for example, in aqueous medium buffered with PBS (pH7.2). The modification reaction is, for example, carried out for 6 hoursat ambient temperature (25° C.) and then for 12 hours at 4° C. withgentle shaking. ANHS is sparingly soluble in water, therefore asolubility enhancing compound such as, by way of example, and notlimitation, dimethyl sulfoxide may, for example, and without limitation,be used to facilitate the reaction between activated acid groups andprimary amine groups on the tissue. In place of dimethyl sulfoxide,other solvents may also be used. Water soluble solvents are morepreferred. Preferred solvents include but not limited to ethanol,methanol, isopropanol, n-methyl pyrrolidinone, dimethyl acetamide,dimethyl formamide, tetrahydrofuran, dixoane, acetone, methyl ethylketone and the like. The reaction occurs in mild conditions, in water(PBS, pH 7.2) and is usually complete in about 24 hours, more preferablywithin about six hours. Acidic or alkaline pH conditions may be used toalter reaction kinetics, but mild reaction conditions are generallypreferred. The reaction may be carried out in pH ranging from 9 to 6,more preferably 6 to 8 and even more preferably at pH 6.5 to 7.5. Manybuffering agents may be used to control the pH. Sodium bicarbonatebuffer may be used to achieve pH around 9, phosphate buffer, triethanolamine buffer, 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid Sodiumsalt (MES) buffer, 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid(HEPES) buffer and the like may be used to control the pH around 7.Acetate buffer may be used to maintain the pH around 4. The tissuemodification can also be substantially completed in about 10 minutes andtherefore may permit the use of this method during a surgical procedureto modify an autologous tissue. In another embodiment of this method,the modification reaction is carried out under mild acidic conditions;for example, without limitation, a MES buffer, (pH 6.5). In this case,the ANHS is added every 1.5 hours for example, without limitation, toreplace the hydrolyzed ANHS. ANHS is generally deactivated by reactionwith water generating acrylic acid. The NHS groups of ANHS react withprimary amines groups of the tissue forming stable amide bonds. Inanother embodiment, the modification reaction is carried out, forexample, without limitation, in organic solvent such as, though notlimited to, dimethyl sulfoxide. Tissue is exposed to, for example,without limitation, 0.2% solution methacrylic anhydride and 1.0%triethylamine in dimethyl sulfoxide for 24-48 hours. The treated tissueis then removed and washed, for example, without limitation, with PBS 3times to remove solvents and stored until further use. Triethyl amine isused as a base catalyst in modification reactions. Other organic basecatalyst such as pyridine, trimethyl amine, N,N-diisopropylethylamine(DIPEA) and the like may also be used. The concentration of methacrylicanhydride and activated NHS ester may be used to control the number ofunsaturated groups in the tissue. The concentration of methacrylicanhydride and activated NHS ester during the modification reaction mayvary from 0.01% to 25%, most preferably 0.5% to 5% even more preferably0.2% to 2%. Room temperature reaction conditions (20-25° C.) are mostpreferred. Temperature higher than 60° C. (above shrink temperature oftissue) is least preferred. The ANHS or methacrylic anhydride reactswith primary amines groups on the tissue. The reaction variables suchas, by way of example, and not limitation, time, temperature,concentration, pressure may be controlled in such a way that about 1 toabout 100 percent primary amine groups on the tissue are modified. Morepreferably, about 10 to about 95 percent amine groups are modified, evenmore preferably about 40 to about 95 percent amine groups are modified.FIG. 2 is an exemplary reaction scheme for tissue crosslinking usingacrylic acid n-hydroxysuccinimide (NHS) ester, in accordance with anembodiment of the present invention; and, FIG. 3 is an exemplaryreaction scheme for tissue crosslinking using di- or polyunsaturatedacid n-hydroxysuccinimide (NHS) ester, in accordance with an embodimentof the present invention. Those skilled in the art, in light of theteachings of the present invention, will recognize that many changescould be made to the tissue modification procedure exemplified above andsuch changes are considered to be within the scope of the presentinvention.

In another embodiment, polymerizable groups are introduced usingglycidyl methacrylate as a tissue modification agent (see Example 37,step 1 described below). The glycidyl group can react with a number ofreactive functional groups such as, by way of example, and notlimitation, primary and secondary amines, carboxyl, hydroxyl, and thiolgroups. This reactivity with multiple functional groups permits one tomodify proteins such as, by way of example, and not limitation, collagenand elastin which are major components of some tissue types. In somecases, tissues may possess high concentration of glycosaminoglycans. Anexample of such tissue, without limitation, is cartilage tissue.Glycidyl methacrylate may be especially useful to modify such tissuesrich with glycosaminoglycans such as, by way of example, and notlimitation, hyaluronic acid. Glycidyl methacrylate may also undergotransesterification reaction with hydroxyl groups in the tissue. Manyreaction conditions, such as, by way of example, and not limitation,aqueous or non-aqueous medium, time, temperature, concentration may, forexample, and without limitation, be used to control degree ofsubstitution. In one preferred embodiment, the reaction temperature ismaintained below 55° C., well below shrink temperature of the tissue. Abase catalyst such as, for example, without limitation, triethyl amine,trimethyl amine, pyridine is used to accelerate the reaction. A phasetransfer catalyst such as, for example, without limitation,tetrabutylamine hydrobromide, tetrapropylamine hydrochloride, may alsobe employed to facilitate the substitution. The glycidyl methacrylate orglycidyl acrylate may, for example, and without limitation, be used, forexample, without limitation, with uncrosslinked tissue or crosslinkedtissue such as, for example, without limitation, glutaraldehyde fixedtissue. In one preferred embodiment, 5 pieces of 28 mm pericardialtissues are treated with a 300 ml solution containing 200 ml PBS, 25 mlglycidyl methacrylate and 25 ml triethylamine at 50° C. for 4 hours.Alternatively, the reaction can also be done at room temperature forabout 24 hours. The concentration of glycidyl methacrylate may be variedform 1 ml to 25 ml to control the amount of unsaturated groups in themodified tissue.

In an alternate preferred embodiment, an unsaturated monoaldehyde suchas, for example, without limitation, acrolein or methacrolein is used tomodify the tissue with unsaturated groups. Acrolein is highly soluble inwater and therefore its modification reaction is very similar to, forexample, without limitation, glutaraldehyde fixation chemistry exceptthat no crosslinking occurs. FIG. 4 depicts the reaction scheme fortissue crosslinking using unsaturated aldehydes, in accordance with anembodiment of the present invention. The acrolein reacts with primaryamines groups on the tissue. Reaction variables such as, by way ofexample, and not limitation, time, temperature, concentration, pressureare controlled in such a way that about 1 to about 100 percent primaryamine groups on the tissue are modified. More preferably, about 10 toabout 95 percent amine groups are modified, even more preferably about40 to about 95 percent amine groups are modified. In the presentembodiment, an aqueous reaction medium is preferred for the modificationreaction. Non-aqueous or semi aqueous medium also may, for example, andwithout limitation, be used in some embodiments. The concentration ofacrolein or methacrolein may range from about 0.1% to about 20%, morepreferably about 0.1% to about 5%, even more preferably about 0.1% toabout 2%. To prevent unwanted free radical polymerization of acrolein ormethacrolein, solutions may be supplemented with free radicalpolymerization inhibitors such as, by way of example, and notlimitation, hydroquinone in the reaction mixture. Like glutaraldehyde,acrolein or methacrolein also have antimicrobial properties andtherefore may help in reducing bacterial and viral contamination of thetissue.

Unsaturated acid derivatives such as, by way of example, and notlimitation, methacrylic anhydride, acrylic anhydride, acryloyl chloride,methacryloyl chloride may also be used to introduce unsaturated groupsinto the tissue. Primary amine residues are target of such modificationchemistries. In one embodiment, methacrylic anhydride is used to modifybovine pericardial tissue. The reaction is carried out in basic bufferedmedium at low temperature for several days to achieve the desired degreeof modification. In another embodiment, the reaction is carried out inaprotic solvent such as, by way of example, and not limitation,dimethylsulfoxide (DMSO) in presence of triethylamine (TEA) as a basecatalyst. Five 1 cm by 1 cm pieces are treated with 10 ml DMSOcontaining 0.02 ml methacrylic anhydride and 0.2 ml TEA. The reactionwas carried out at ambient temperature (30)° for 24-48 hours

In many of the modification chemistries discussed above, the tissuemodification may be carried out in aqueous medium or non-aqueous mediumsuch as, by way of example, and not limitation, organic solvent. Thepreferred organic solvents are commonly used organic solvents dimethylsulfoxide, n-methylpyrrolidinone, ethanol and the like. However, otherorganic solvents may also be employed. Reaction conditions such as, byway of example, and not limitation, time, temperature, concentrationwill depend on the degree of substitution desired.

The unsaturated group modification methods, which are taught by way ofexample in the present detailed description, may be modified to obtain adesired degree of substitution. Synthetic methods as such, andotherwise, will be apparent to those skilled in the art, in light of theteachings of the present invention. In most cases, up to about 70 toabout 100 percent modification of primary amine groups is preferred tosuppress the immune response and to be useful in the crosslinkingreactions motioned below.

The unsaturated group incorporation methods for tissues as discussedabove may also be used to modify collagen solid particles or collagensponges or fibers in the solid state. For example, collagen spongesobtained from commercial vendors like Kensey Nash may be treated withANHS solution or with methacrylic anhydride in DMSO to incorporateacrylic/methacrylic groups in the collagen sponge. The unsaturated groupmodified collagen sponge is then crosslinked using methods similar tothe tissue crosslinking methods mentioned in this document. Thecrosslinked collagen sponge may have a higher degradation time ascompared to uncrosslinked sponge and may be useful in tissue engineeringapplications especially in bone tissue engineering applications. Thecrosslinked collagen may be incorporated with BMP to accelerate the boneformation. Though not required, the BMP may be added prior tocrosslinking and polymerization reaction.

The first step shown by way of example in the FIG. 1 for thecrosslinking of unsaturated group modified tissue will next be describedin some detail. In the second step, the unsaturated groups introduced bytissue modification methodologies discussed above are used in tissuecrosslinking. In one approach, the unsaturated groups are dimerized,polymerized, or copolymerized and crosslinked with monomers that canundergo free radical polymerization or cyclic dimerization. In anotherapproach, the unsaturated groups are reacted with di- or polyfunctionalmercapto compounds to introduce the crosslinking in the tissue.

Crosslinking of Unsaturated Groups in the Tissue Using Free RadicalCrosslinking

Another aspect of the present invention is directed to a method ofcrosslinking a tissue, including treating the unsaturated group modifiedtissue under effective crosslinking conditions with a free radicalcrosslinking mechanism. In one embodiment of the present invention, amethod for cross-linking collagen-containing biological tissue undereffective conditions wherein at least a portion of the tissue functionalgroups form a covalent bond with a compound having the formula F—X—U,wherein F, X, and U are as defined above, then treating the tissue undereffective crosslinking conditions with a compound having at least onefree radical polymerizable group. Accordingly, the tissue used in thistype of crosslinking is first modified using unsaturated groups asdiscussed above. More preferably, the tissue modified with unsaturatedgroups such as, by way of example, and not limitation, acrylamide,methacrylamide, acrylate or methacrylate group is used in thecrosslinking reaction. In one embodiment of the present invention, themethod of cross-linking the tissue is achieved by steps that include,without limitation, treating unsaturated group modified tissue undereffective cross-linking conditions with a free radical polymerizablemonomer.

In one embodiment, unsaturated group modified tissue is graduallydehydrated in series of aqueous alcohol solutions. The dehydration mayalso be done using other techniques such as, by way of example, and notlimitation, lyophilization or vacuum drying and the like. The dehydratedtissue is then exposed to or incubated in a monomer/monomers solution(n-vinyl pyrrolidinone as an example) containing free radicalphotoinitiator. The incubation is carried out until the infusion ofmonomer/initiator is complete throughout the tissue matrix. This mayrequire incubation from minutes to few days. More preferably, tissue isincubated for about 0.1 minute to about 16 hours. Even more preferably,for about 5 minutes to about 2 hours. The monomer/photoinitiator-soakedtissue is then exposed to long wavelength ultraviolet light untilpolymerization and crosslinking is achieved. The photoinitiator inpresence of UV light initiates polymerization of monomer. Theunsaturated groups on the tissue copolymerize with the monomer andcrosslink the tissue. The overall tissue crosslinking reaction is shownin the example of FIG. 1. In order to effectively crosslink the tissue,the growing polymer free radical must copolymerize or react withunsaturated groups on two collagen chains. This is achieved, forexample, without limitation, by selecting a number of polymerizationvariables such as, by way of example, and not limitation, monomerconcentration, percent acrylate modification of the tissue, initiatorconcentration, UV light intensity, temperature, and the like. In onepreferred embodiment, acrylic acid succinimide ester modified orglycidyl methacrylate tissue is dehydrated in aqueous ethanol solutions.The ethanol-treated tissue is then transferred to n-vinyl pyrrolidinone(NVP) solution containing Irgacure 2959[(4-(2-hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone)] as a longwavelength ultraviolet photoinitiator. The NVP-soaked tissue isirradiated up to 5 minutes using long wavelength UV light (Black-Ray UVlamp, 360 nm flood light, 10000 mW/cm2 intensity). The irradiated tissueis washed with PBS solution to remove unpolymerized monomer andpolymerized but uncrosslinked soluble polyvinyl pyrrolidinone polymer.

Glycidyl methacrylate tissue incubated in n-vinyl pyrrolidinone solutionbut not exposed to light is referred to as the dark control. Untreatedtissue is referred as control tissue. Table 1 gives pepsin digestiondata on pericardial tissue fixed/crosslinked using glycidylmethacrylate/n-vinyl pyrrolidinone or glycidyl methacrylate/acrylicacid. The data of Table 1 indicates that the untreated control tissueand dark control tissue degraded completely in a pepsin solutionindicating their susceptibility to enzymatic degradation. The tissuetreated with glycidyl methacrylate and subsequently treated with n-vinylpyrrolidinone or acrylic acid monomer and free radical initiation showedless than 9 percent weight loss when exposed to pepsin solutionindicating their high stability towards enzymatic degradation. Thesource of the tissue such as, by way of example, and not limitation,bovine pericardium or sheep pericardium did not show a significantdifference in weight loss indicating the applicability of this treatmentto tissues from different animals. Table 2 lists shrink temperaturevalues for control and treated tissue. Again, the n-vinylpyrrolidinone-treated tissue showed a significantly higher shrinktemperature as compared to untreated control tissue indicating tissuestability and crosslinking.

TABLE 1 Pepsin digestion data for sheep and bovine pericardium tissuefixed using glycidyl methacrylate and free radical polymerizablemonomer. Pepsin Digestion Tissue Treatment Weight Loss (%) Controltissue (untreated tissue) 100*   Dark control (all treatments exceptexposure 100*   to light) Crosslinked sheep tissue (exposure to UV light8.3 and photoinitiator, monomer acrylic acid) Crosslinked sheep tissue(exposure to UV light 6.5 and photoinitiator, n-vinyl pyrrolidinone)Crosslinked bovine tissue (exposure to UV light 2.5 and photoinitiatorand n-vinyl pyrrolidinone) *The tissue is totally disintegrated andcould not be washed and weighed.

TABLE 2 Shrink Temperature data for control and crosslinked tissueShrink Temperature Tissue treatment measured by DSC (° C.) Untreatedcontrol sheep tissue 60.9 Sheep pericardium tissue fixed using 82.1glycidyl methacrylate and n-vinyl pyrrolidinone

The crosslinked tissue exhibited a different texture to a human hand ascompared to uncrosslinked tissue indicating crosslinking. Thecrosslinked tissue showed substantially higher shrink temperature ascompared to uncrosslinked tissue indicating crosslinking formation.Unlike glutaraldehyde tissue, the crosslinked tissue is found to benon-cytotoxic. In another embodiment of the present invention, a thermalinitiator such as, by way of example, and not limitation,azobisisobutyronitrile is used to initiate polymerization andcrosslinking of tissue. Yet in another embodiment, radiation or electronbeam irradiation or short UV is used to polymerize and crosslink thetissue in presence NVP as comonomer. In these systems, no initiatorneeds to be added. In some embodiments, a free radical photoinitiatormay be chemically or physically attached to tissue being fixed. Oneaspect of such treatment is that the solution can be fixed withoutexternal free radical polymerization initiator.

Those skilled in the art of free radical polymerization chemistry willunderstand that many changes could be made in the polymerization andcrosslinking of tissue, in light of the teachings of the presentinvention. The polymerization and crosslinking could be initiated usinga number of monomers. For example, many monofunctional monomers whichform linear soluble polymers could be used in copolymerization andcrosslinking of tissue and these include, but not limited to, acrylicand methacrylic esters such as, by way of example, and not limitation,methyl methacrylate, 1-Adamantyl methacrylate, 1-allyloxy-2,3-propanediol, ethyl methacrylate, dimethylaminoneopentyl acrylate, propylmethacrylate, isopropyl methacrylate, n-acryloyl sarcosine methyl ester,dimethylaminoethyl methacrylate, hexyl methacrylate, butyl methacrylate,dicyclopentenyl acrylate, 2-ethyl hexyl methacrylate,2-methacryloyloxyethyl phosphorylcholine, ethoxyethyl methacrylate,octyl methacrylate, 2-hydroxy-4-acryloylethoxy benzophenone, stearylmethacrylate, 2-hydroxyethyl methacrylate, 2-aminoethyl methacrylate,glycidyl methacrylate, isocyanatoethyl methacrylate, cyclohexylmethacrylate, methyl acrylate, ethyl acrylate, propyl acrylate,isopropyl acrylate, hexyl acrylate, butyl acrylate, 2-ethyl hexylacrylate, octyl acrylate, stearyl acrylate, 2-hydroxyethyl acrylate,glycidyl acrylate, isocyanatoethyl acrylate, ethoxyethyl acrylate,cyclohexyl acrylate, 2,2,3,4,4,4-hexafluorobutyl acrylate;2,2,3,4,4,4-hexafluorobutyl methacrylate; 2,2,3,3,3-Pentafluoropropylacrylate; 2,2,3,3,3-Pentafluoropropyl methacrylate; trifluoroethylmethacrylate, trifluoroethyl acrylate; tetrafluoroethylene, fluorinatedvinyl ethers, alkyl acrylamides and methacrylamides such as, by way ofexample, and not limitation, acrylamide, methacrylamide, n-propylacrylamide, butyl acrylamide, isobutyl acrylamide, ethyl acrylamide,tertiarybutyl acrylamide, n-propyl methacrylamide, butyl methacrylamide,isobutyl methacrylamide, ethyl methacrylamide, tertiarybutylmethacrylamide, pentyl methacrylamide; pentyl acrylamide; styrene andstyrene derivatives such as, by way of example, and not limitation,methyl styrene, chlorostyrene; vinyl ethers such as, by way of example,and not limitation, glycidyl vinyl ether; acrylonitrile, acrylic acid,methacrylic acid, allyl alcohol, allyl amine, polysiloxane or polyetherbased macromonomers, polyurethane acrylates and methacrylate and thelike. Many monofunctional monomers which form linear soluble polymerscould be used in copolymerization and crosslinking of the tissue andthese include, but not limited to, n-vinyl pyrrolidinone, acrylic acid,2-hydroxyethyl methacrylate, glyceryl methacrylate, polyethylene glycolacrylate, 2-hydroxypropyl acrylate, monomers used in commercial soft andhard contact lens manufacturing, 2-hydroxypropyl methacrylate,acrylamide, vinyl acetate, silicone-based acrylates and methacrylates,silicone-based monomers used on contact lens manufacturing, n-isopropylmethacrylate, glycidyl methacrylate, glycidyl acrylate, methacrylicacid, acrylonitrile, styrene, methyl methacrylate, methyl acrylate, PEGor PEO acrylates and methacrylates, monomers obtained from fatty acidand fatty acid derivatives, monomers obtained from low molecular weightpolyethylene, polypropylene other vinyl polymers and the like. Monomerswhich are soluble or substantially soluble in water (solubility greaterthan 1 percent in water) are preferred. The polymerization andcrosslinking reaction can be performed in aqueous, semiaqueous ornon-aqueous medium. In general, water or alcohol water mixtures arepreferred. In water, extreme high or low pH with prolonged exposuretimes, typically greater than 1 hour are not preferred at least becausesuch conditions can damage or denature the tissue. The preferred pHrange is between about 10 to about 2; pH range between about 9 to about6 is even more preferred, and physiologic pH range around about 7.2 ismost preferred. Many buffers can be used to control the pH. Buffers suchas, by way of example, and not limitation, phosphate buffer, triethanolamine buffer, acetate buffer, borate buffer, bicarbonate buffer, HEPESbuffer are preferred. Among non aqueous media, monomers that can be usedas solvents are preferred. For example, n-vinyl pyrrolidinone is aliquid and can solubilize a variety of other monomers withoutsignificantly affecting the tissue. Other preferred liquid monomersinclude liquid PEG-based monomers, n-vinyl caprolactum, 2-hydroxyethylmethacrylate, glyceryl methacrylate, and glycidyl methacrylate. organicsolvents such as, by way of example, and not limitation, n-methylpyrrolidinone, dimethylsulfoxide, dimethylformamide. dimethylacetamideetc.; lower ketones (i.e., having from about 3 to 6 carbons) such as, byway of example, and not limitation, methyl ethyl ketone orcyclohexanone; and polyhydroxy compounds such as, by way of example, andnot limitation, glycerol, ethylene glycol, or polyethylene glycolshaving molecular weights less than about 1000 also could be used forpolymerization and crosslinking. The free radical initiation necessaryfor polymerization could also be achieved by exposing the tissue togamma radiation, electron beam radiation, UV radiation, usingphotoinitiator or thermal initiators. The gamma radiation, electron beamirradiation or short UV irradiation may not need external free radicalinitiator. The photoinitiator could be selected depending on thewavelength of interest. For long wavelength UV light, initiator Irgacure2959, Irgacure 651 (2,2-dimethoxy-2-phenyl acetophenone), benzophenoneand the like could be used. For visible light initiation, initiatingsystems eosin-triethanol amine (irradiation wavelength 512 nm),methylene blue-triethanol amine system (irradiation wavelength 632 nm)and the like could be used. The choice of various photoinitiators andtheir corresponding wavelength can be found in photopolymerizationliterature art. Preferred photopolymerization conditions,photoinitiators are described in U.S. Pat. No. 5,410,016 (Hubbell, etal), which is cited here for reference only as one suitable technique.There are many free radical polymerization systems that are known in thedental cement art, contact lens manufacturing art, or in theradiation-cured coating art which can be used in polymerization orcrosslinking of unsaturated group modified tissue. However, initiatingsystems based on long UV or visible light initiated systems arepreferred due to higher penetration of light inside the tissue. Ingeneral, the higher the wavelength light, the greater penetration inside the tissue. The penetration of light into the tissue can also becontrolled by controlling photoinitiator concentration or a chromophorethat selectively absorbs light from irradiation sources. The amount offree radical initiator in the formulation may range from about 0.01% toabout 2 percent relative to the weight of monomer. Most preferably, thepreferred initiator amount is about 0.05% to about 1%. Various lightsources such as, by way of example, and not limitation, xenon lamp,mercury lamp, halogen lamp, argon ion laser, solid sate semiconductorbased lasers may, for example, and without limitation, be used forphotopolymerization and crosslinking of tissues. The light used in thephotopolymerization could be transported using fiber optic or liquidguide as known in the photochemistry art or surgical laserinstrumentation art. Various types of thermal initiators could also beused; these include, but not limited to, azo-based initiators such as,by way of example, and not limitation, azobisisobutyronitrile,peroxide-based initiators such as, by way of example, and notlimitation, benzoyl peroxide, inorganic initiators such as, by way ofexample, and not limitation, potassium persulfate or ammonium persulfateand the like. The polymerization medium could be aqueous or non-aqueous.In one embodiment, the tissue is dehydrated by lyophilization and thenincubated with an aqueous solution of polyethylene glycol acrylate. Thepolyethylene glycol acrylate is then photopolymerized and crosslinked inwater in presence of unsaturated group modified tissue.

The polymerization and crosslinking may also be conducted in water undermild conditions. For example, without limitation, unsaturated groupmodified tissue is incubated in a 50% aqueous solution of acrylamide or2-hydroxyethyl methacrylate containing 2% Irgacure 2959 for 24 hours.Acrylamide concentration could be varied from about 10% to about 60% inwater with 0.1% Irgacure 2959. The incubated tissue is removed andexposed to long UV light for 10 minutes to polymerize and crosslink thetissue. The unreacted monomers are removed by washing. In oneembodiment, glycidyl methacrylate modified bovine pericardial tissue(see Example 37 described below) was exposed to with 10-50% acrylamidesolution containing 0.1% Irgacure 2959 for 30 minutes and then exposedto long wavelength UV lamp (360 nm light) for 5 minutes. The resultanttissue showed significantly higher shrink temperature as compared tountreated pericardial tissue.

The degree of polymerization and crosslinking can also be controlled byadding special additives known in the free radical polymerization art.For example, a chain transfer agent such as, by way of example, and notlimitation, mercapto ethanol may be added to control the molecularweight of polymer formed in the tissue. Other chain transfer agents thatcan be used include, but are not limited to, biocompatible thiol,bromine or chlorine containing organic compounds. These compoundsinclude, but are not limited to cysteine (amino acid), carbontetrachloride, chloroform and the like. Inhibitor may be added tomonomers to prevent unwanted polymerization during storage. The monomermay be purged with inert gas to remove dissolved oxygen. The molecularweight of polymer produced may range from 1000 to 5 million g/mole, morepreferably 5000 to 150000 g/mole, even more preferably 10000 to 10000g/mol. The choice of molecular weight will depend on the intended use.Typically tissue-polymer composite which require superior mechanicalproperties may use high molecular weight polymer (>10000 g/mole).

Polymeric crosslinks formed by free radical polymerization generallyresults into polymers with verging degree of molecular weights or chainlengths. The molecular weight distribution of polymer will depend onexperimental conditions used. Typically, the molecular weightdistribution, which is generally defined as ratio of weight averagemolecular weight (Mw) to number average molecular weight (Mn), is 1.1 to4, more typically 1.3 to 2.5 for most polymeric systems formed by freeradical polymerization. Since polymer formed during crosslinkingreaction as described above have a range of molecular weight, thepolymeric chain length may also vary. Thus, this invention overcomes thelimitation of fixed molecular length crosslinking methods such asglutaraldehyde crosslinking and forms polymeric crosslinks with variouslengths or molecular weights.

Monomers used may be purified before using them in the polymerizationand crosslinking reaction. The purification may be achieved bytechniques such as, by way of example, and not limitation, vacuumdistillation, column chromatography, recrystallization and the like.Oxygen in the atmosphere may act as an inhibitor and can alterpolymerization kinetics. The effect of oxygen can be minimized byconducting the polymerization and crosslinking under inter gasatmosphere. Inert gases such as, by way of example, and not limitation,nitrogen, carbon dioxide, and argon are most preferred.

In one embodiment, the tissue is first crosslinked using a crosslinkercontaining two or more tissue reactive groups and a polymerizable doublebond. The double bond in the crosslinker is then further crosslinkedusing free radical polymerization. One aspect of this type ofcrosslinking is that two methods of crosslinking are used in preparingthe crosslinked tissue. For example, tissue may be first crosslinked bydi- or polyfunctional unsaturated compounds such as, by way of example,and not limitation, n,n-methylenebisacrylamide using a Michael additionreaction known in the synthetic organic chemistry art or a derivative ofunsaturated di- or polyacid. This type of crosslinking is similar toglutaraldehyde crosslinking known in the art. The dangling or unreacteddouble bonds in the crosslinking reaction and double bonds in thecrosslinks may be copolymerized and crosslinked using free radicalphotopolymerization as described earlier. This concept further isfurther illustrated by way of example, and not limitation, in FIG. 3using unsaturated di-polyacid derivative as an example. The preferreddi- or polyunsaturated compounds that may be used include, but are notlimited to, n,n-methylenebisacrylamide, polyethylene glycol diacrylate,polyethylene glycol dimethaacrylate, trimethylol propane triacrylate,trimethylol propane trimethacrylate; dipentaerythritol methacrylate, andthe like. In one exemplary approach, the tissue is first crosslinked ormodified with di- or polyunsaturated diacids (see FIG. 3). These diacidsinclude, but are not limited to, fumaric acid, itaconic acid, maleicacid, castor oil, and the like. Unsaturated acids such as, by way ofexample, and not limitation, fumaric acid, maleic acid and itaconic acidare preferred due to their high reactivity and ability to copolymerizewith variety of monomers. The di- or polyunsaturated acids could beincorporated into the tissue using several chemical methods known in theprotein modification art. For example, unsaturated acids like fumaricacid could be incorporated by incubating the tissue in a 1% solution offumaric acid in PBS or MEM buffer at about pH 4.0 to about 8.0 in thepresence of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Hydrochloride(EDC) as an esterification catalyst (2%) and n-hydroxy succinimide asco-catalyst (2%). In a preferred method (see Example 32 describedbelow), an activated derivative of fumaric acid is prepared separately.The preferred derivative is n-hydroxysuccinimide ester (NHS) orn-hydroxysulfosuccinimide ester (SNHS) or fumaryl chloride. The SNHSester is soluble in water and does not need organic solvents to disperseit. Tissue such as, by way of example, and not limitation, pericardialtissue is first reacted with fumaric acid NHS derivative in PBS. Themodified tissue shows increased shrink temperature as compared tounmodified tissue indicating crosslinking. The tissue is also lesscytotoxic or non-cytotoxic when compared with tissue fixed using 0.4%glutaraldehyde. The fumaric acid-modified tissue is further reacted orcrosslinked with monomers capable of undergoing free radicalpolymerization as mentioned previously. In one exemplary approach,fumaric acid-modified tissue is reacted or copolymerized with n-vinylpyrrolidinone using UV photopolymerization. The polymerization reactionvariables as well as types of monomers copolymerized can be changed toobtain a desirable implantable tissue material. Bovine pericardialtissue modified with fumaryl chloride and crosslinked withn-hydroxysuccinimide showed higher shrink temperature as compared tounmodified tissue indicating crosslinking.

Yet another embodiment of the present invention provides for a methodfor making radio-opaque implantable tissue including the steps ofproviding a tissue suitable for human implantation; exposing the tissueto fluid including a radio-opaque compound capable of reacting with atissue; and covalently bonding a radio-opaque compound to the tissue.Monomers containing radio-opaque atoms such as, by way of example, andnot limitation, iodine or metal ions are used for polymerization andcrosslinking. The incorporation of radio-opaque polymerizable monomerswill provide radio-opacity to the tissue. The preferred monomers thatmay, for example, and without limitation, be used are polymerizablederivatives or iodinated compounds such as, by way of example, and notlimitation, polymerizable monomer obtained by esterification oftriiodobenzoic acid and 2-hydroxyethyl methacrylate. Other iodinatedderivatives that may be converted into polymerizable monomers byesterification of hydroxyl groups include, but are not limited toiohexyl, metrizamide, iopamidol, iopentol, iopromide, erythrosin, andioversol. The polymerizable derivative of metrizamide is most preferred.The radio-opacity is controlled by the changing the amount ofradio-opaque monomer incorporated in the tissue. The iodine content oftissue may vary from about 20 to about 300 mg iodine per gram of tissueweight. More preferably, the amount of iodine may be about 40 to about200 mg/g of tissue.

Tissue could be chemically modified with unsaturated group and freeradical initiating group such as, by way of example, and not limitation,eosin or benzophenone. For example, eosin could be physically adsorbedor chemically bound to the tissue using the esterification of carboxylgroup on the eosin molecule. Such initiator and unsaturated groupmodified tissue can be crosslinked using free radical initialingchemistry such as, by way of example, and not limitation,photoinitiation chemistry as discussed previously. In a similar manner,tissue could be chemically modified with thermal initiator andpolymerizable group on the same protein chain. One aspect of suchsystems is that no external free radical initiator is needed to initiatefree radical polymerization and crosslinking.

In some embodiments, the tissue could be modified with chemical groupswhich undergo cyclic dimerization upon exposure to heat or light energycould be attached to tissue and then subsequently dimerized to crosslinkthe tissue. The compounds which undergo cyclic dimerization include, butare not limited to, cinnamic acid, coumarin, chalcone and thymine Manyfunctional groups which undergo cyclic dimerization are known in thephotochemistry art and could be used; however the derivatives ofcinnamic acid are most preferred. Cinnamic acid and its derivatives areknown to undergo photodimerization on irradiation with ultravioletlight. This reaction is highly specific and does not require freeradical photoinitiator. It can be carried out in liquid, solid, orsolution state. Photocurable polymers which can be cured or crosslinkedby photodimerization reaction of cinnamic acid have been extensivelyused in arts related to lithography, paints and printing. In a preferredembodiment, the tissue is first modified with cinnamic acid derivativesuch as, by way of example, and not limitation,n-hydroxysulfosuccinimide derivative of cinnamic acid or cinnamoylchloride. The modified tissue is then exposed to long wavelengthultraviolet light (360 nm) or visible light to dimerize and crosslinkthe unsaturated groups in the tissue.

Prosthetic Tissue with Biostable and Biodegradable Regions

In some embodiments, photopolymerization and crosslinking is controlledby the exposure of light to certain parts of the tissue. This may, forexample, and without limitation, be used to crosslink only selectedparts of the tissue. In one exemplary embodiment, the tissue was exposedto the light through a mask which permits exposure of light only on aselected predetermined area. The tissue exposed to the light undergoespolymerization and crosslinking. Parts of the tissue, which are notexposed to light remain uncrosslinked and therefore remain susceptiblefor degradation. This concept is illustrated, by way of example, and notlimitation, in FIG. 5. In one exemplary embodiment (see Example 40described below), a 2 cm diameter tissue treated with glycidylmethacrylate as shown in Example 37 part 1. The tissue is incubated in,by way of example, and not limitation, n-vinyl pyrrolidinone solutioncontaining photoinitiator (Example 37, part 2). The incubated tissue iskept on a glass plate. Two 4 mm diameter aluminum foil sections or arrowshape foil sections are cut and placed on the tissue. The tissue is thenexposed to long wavelength UV light for, by way of example, and notlimitation, 5 minutes for polymerization and crosslinking of the tissue.The same procedure is repeated from other side of the tissue. The tissueis then exposed to pepsin solution. The areas of the tissues which arecovered by aluminum foil showed substantial degradation and iscompletely solubilized while the light-exposed tissue did not show signsof degradation and was mechanically intact. Thus a pattern of degradableand non-degradable regions is created in the same tissue. Many types ofpatterns of crosslinked and non-crosslinked tissue may be generatedwhich include rectangular, circular or complex patterns or shapes insidethe tissue matrix using this approach. The selective crosslinking andpolymerization approach could be, for example, used to selectivelyreinforce certain parts of medical devices such as, by way of example,and not limitation, heart valve. Certain high-stressed sections ofbovine pericardial heart valve tissue are susceptible to calcificationand mechanical failure. Such sections may be selectively crosslinked byexposing those sections to light. It can also be used in vivo tissueengineering application.

In some embodiments, the solvents and other crosslinking constituentsused during the polymerization and crosslinking process may be subjectedto mild or high pressure to obtain a suitable crosslinking density.

Shape Preserving Tissue Fixation

In some preferred embodiments, the collagen fibers may be strained ororiented along the direction of pull prior to polymerization andcrosslinking. The tissue orientation may be, for example, and not by wayof limitation, achieved through the use of mechanical force. Forexample, unsaturated group modified bovine pericardial tissue is dried,infused with n-methyl pyrrolidinone and photoinitiator 2959 and thenstretched to about 110 to about 150% of its length to orient the fibersalong the direction of force. The monomer and initiator infusion couldalso be made after orienting the tissue. The strained tissue is thenexposed to long UV light for polymerization and crosslinking. Thepolymerization and crosslinking will “lock in place” the orientedstructure of the proteins/fibers in the tissue and thus yielding atissue with superior mechanical properties, especially along the axis oforientation. Modification of various variables used in this method suchas, by way of example, and not limitation, direction of force applied,axis of orientation, % stretching or compression used, polymerizationconditions, monomers used must be optimized for a given application. Itwas discovered that some monomers which produced highly crosslinked andrigid polymers can substantially preserve the shape of a crosslinkedtissue. This concept is schematically illustrated, by way of example,and not limitation, in FIG. 6. In one exemplary embodiment (see Example29 described below), a 15 cm long and 3 mm wide strip of bovinepericardial tissue modified with glycidyl methacrylate was infused withtriethyleneglycol dimetharylate or tetraethyleneglycol dimethacrylatemonomer containing 500 ppm Irgacure 651 photoinitiator. The monomerinfused strip is then spirally wound, by way of example, and notlimitation, on a 5 mm diameter mandrel (glass rod) in a helical shape.The tissue was then exposed to long UV light for 5 minutes (Black-Ray UVlamp, 360 nm light, 10000 mW/cm2 intensity) for polymerization andcrosslinking of the monomer. The glass rod was removed. The tissue shapeof helical coil was substantially preserved after removal of glass rodor mandrel. When coiled tissue was stretched along the axis of thehelical coil, and the stress is removed, the tissue substantiallyreturned to the helical coil shape indicating shape memory property.This ability to remember the shape after fixation on crosslinking can bebeneficial in making novel medical devices. For example, it can be usedto form a tissue-based stent-like devices. The light exposure andpolymerization can be done in situ at a surgical site. The monomer andphotoinitiator infused tissue strip is immobilized, by way of example,and not limitation, on an angioplasty catheter. The catheter is capableof emitting 360 nm light carried using, by way of example, and notlimitation, fiber optic cable on the balloon surface. The tissue istransported at the angioplasty site using, for example, withoutlimitation, standard balloon angioplasty techniques, expanded andexposed to emitting 360 nm light until polymerization is complete(typically less than 5 minutes). The balloon is withdrawn and coiledtissue is left at the angioplasty site which provides mechanical supportsimilar to metal-like stent. In another modification of this approach, amonomer infused unsaturated group modified pericardial tissue asmentioned previously was converted or sewn into a 6 mm diameter tube.The tube was then mounted on a 6.1 mm diameter stainless steel mandreland compressed to reduce the length of the tube (10 to 50 percentreduction in length). The compressed tissue is then exposed to UV lightto crosslink the monomer and fix the tissue. The shape of the tissue ispreserved in a compressed shape. The mandrel is removed and thecompressed tissue tube held the shape with accordion-like wrinkles inthe tube. The tube is compressible along the axis of the tube. Such atube may be useful to make peripheral vascular and coronary graft andother medical devices. Any desired drug such as, by way of example, andnot limitation, heparin, by way of example and not limitation, may beincorporated in the tube to influence the desired biological ortherapeutic outcome.

One embodiment of the present invention provides for a method for makinga tissue capable of remembering a shape.

In one embodiment, a radiation polymerization or electron beam-inducedpolymerization technique is used to form a crosslinked tissue. Such areaction can be carried out in the solid state. The gamma radiation orelectron beam irradiation generates several free radicals in the tissue.The generated free radicals react with unsaturated groups in the tissueto promote crosslinking. Radiation polymerization may not require freeradical initiators. The dose of radiation will depend on the amount ofpolymerization and crosslinking desired. It is contemplated that suchoptimization can be readily undertaken by one skilled in the art, inlight of the teachings of the present invention, without undueexperimentation. Low to moderate radiation doses, typically below about3-5 MRAD, are preferred at least because high radiation dose may alsodegrade collagen molecular chain.

One aspect of the present invention is that many monomers with differentproperties such as, by way of example, and not limitation, chargedensity, hydrophobicity, thermosensitivity could be used to impartdifferent properties to the crosslinked tissue. In one embodiment, afunctional monomer such as, by way of example, and not limitation,glycidyl methacrylate which has a glycidyl group is copolymerized alongwith vinyl pyrrolidinone. The polymerization of glycidyl methacrylateprovides glycidyl functional groups on the crosslinked polymer chainwhich could be further reacted using standard epoxy chemistry foradditional crosslinking or other types of tissue modifications.Functional monomers that could be used, but are not limited to, are2-isocyanate methacrylate, acrylic acid, methacrylic acid,2-hydroxyethyl methacrylate, glyceryl methacrylate and the like. Asthose skilled in the art will appreciate, chemical variables such astime, temperature, concentrations, pressure, catalysts and the like maybe controlled to promote additional crosslinking. Moreover, the use ofthese variables will, at least, depend on a particular chemical reactionused in crosslinking.

In another embodiment, monomers containing long alkyl chains are used inpolymerization and crosslinking. The introduction of a long chainthrough crosslinking can reduce its calcification potential and mayimprove hemocompatibility of the crosslinked tissue. It is hypothesizedthat long chain alkyl chains, typically more than about 8 carbons (C8),more typically with carbon lengths such as, by way of example, and notlimitation, C16-C18, specifically bind to albumin and thus improvehemocompatibility of the tissue. However, shorter alkyl chains,typically >C4, may also be advantageous for some applications.

In some embodiments, the monomers that give fluorinated polymers uponpolymerization may, for example, and without limitation, be used toincorporate fluorinated polymers in the tissue. The monomers includingfluorinated monomers such as, by way of example, and not limitation,acrylates and methacrylates with fluorinated alkyl chains may, forexample, and without limitation, be used. The fluorinated monomerinclude, but are not limited to, 2,2,3,4,4,4-hexafluorobutyl acrylate;2,2,3,4,4,4-hexafluorobutyl methacrylate; 2,2,3,3,3-pentafluoropropylacrylate; 2,2,3,3,3-pentafluoropropyl methacrylate; trifluoroethylmethacrylate, trifluoroethyl acrylate, tetrafluoroethylene and the like.

In another embodiment of the present invention, a silicone- orpolydimethylsiloxane-based monomer or macromonomer is used inpolymerization and crosslinking of the tissue. Many silicone- orpolydimethylsiloxane-based monomers could be suitably used and are knownto one skilled in the art. The incorporation of highly elastomericsilicone-based monomers incorporates silicone rubber in the tissuematrix which provides elastomeric properties and improves oxygenpermeability of the tissue in many applications. Polypropylene glycoland some polyurethane acrylates and methacrylate-based monomers thatalso form elastomeric polymeric crosslinks may also be used.Commercially available polydimethyl siloxane-based macromonomers knownin the soft contact lens manufacturing art can be useful in manyapplications, at least because use of such monomers is alreadycommercialized in high oxygen permeable contact lens applications.

In another exemplary embodiment (see Example 11 described below), amonomer which produces a thermosensitive polymer such as, by way ofexample, and not limitation, poly(n-isopropyl acrylamide) is used inpolymerization and crosslinking of tissue. The incorporation ofthermosensitive polymer crosslinked network permits one to rehydrate thecollagen chains at low temperature (<10° C.). At body temperature (37°C.), poly(n-isopropyl acrylamide) becomes hydrophobic. The hydrophobicdomains thus formed may provide interesting properties to the tissue.For example, hydrophobic domains may, for example, and withoutlimitation, be used to lock or dissolve hydrophobic drugs in the treatedtissue. The hydrophobic domains may also provide additional protectionagainst enzymatic degradation of the tissue. Many n-alkylacrylamide-based monomers which upon polymerization yieldthermosensitive polymers could be used, these include, but are notlimited to, n-ethyl acrylamide, n-propyl acrylamide, n-isopropylacrylamide, n-butyl acrylamide, n-isobutyl acrylamide, n-tertiarybutylacrylamide, n-pentyl acrylamide, and the like. In one illustrativeembodiment, glycidyl methacrylate treated pericardial tissue (seeExample 37, part 1 described below) is exposed to, for example, withoutlimitation, 50 percent n-isopropylacrylamide solution in watercontaining 0.1% Irgacure 2959 for 2 hours. The monomer infused tissuewas exposed to long UV light for 5 minutes (Black-Ray UV lamp, 360 nmlight, 10000 mW/cm2 intensity) for polymerization and crosslinking ofthe monomer. The tissue was washed with distilled cold water to removemonomer and polymerized but not covalently bound polymer. The tissuerepresents an example of pericardial tissue crosslinked usingthermosensitive polymer. The tissue showed substantially higher shrinktemperature as compared to untreated pericardial tissue indicatingcrosslinking using n-isopropylacrylamide. Any number of bioactivecompounds such as, by way of example, and not limitation, paclitaxel,for example, could be diffused inside the matrix at low temperature(typically below 15° in water). At body temperature,n-hydroxysuccinimide becomes hydrophobic trapping the drug in thehydrophobic matrix and releasing it in a controlled manner. Hydrophobicdrugs such as, by way of example, and not limitation, paclitaxel,rapamycin, chlorhexidene gluconate are preferred. Other hydrophobicdrugs may also be used in alternate embodiments of the presentinvention. Certain polymeric monomers or macromonomers, such as, by wayof example, and not limitation, monomer made from block copolymers ofPEG and polypropylene oxide or polypropylene oxide, for examplePEG-PPG-PEG acrylates and methacrylates which are soluble in water atlow temperature may also be used. Alternatively, thermosensitivepolymer-based crosslinkers can also be used to crosslink the tissue. Thecrosslinking may be carried out when the thermosensitive polymercrosslinker is soluble in water.

In another embodiment, the monomers containing charged groups such as,by way of example, and not limitation, sulfonic acid salts or carboxylicacid salts are used for polymerization and crosslinking. It is believedthat incorporation of charged groups in the crosslinked tissue mayreduce the propensity for calcification and improve hemocompatibility.The monomers containing charged groups that may be used, but are notlimited to, are 2-acrylamido-2-methylpropane sulfonic acid,vinylsulfonic acid, styrene sulfonic acid, sulfoethylmethacrylate,sulfopropylmethacrylate, or other vinyl sulfonic acids; acrylic acid,methacrylic acid, fumaric acid, maleic anhydride, maleic acid, fumaricacid, itaconic acid, and the like. Monomers containing sulfonic acidsalts are most preferred. The polymerization of such charged groupscreates localized acidic or basic environment which may reducecalcification upon implantation. In one exemplary embodiment,crosslinking of glycidyl methacrylate modified tissue using2-acrylamido-2-methylpropane sulfonic acid showed substantially highershrink temperature as compared to untreated tissue indicatingcrosslinking.

In another embodiment, the monomers containing free amine groups areused for polymerization and crosslinking of the tissue. The amine groupsin the monomers can be used to introduce additional crosslinking in thetissue. The amine-containing monomers that may be used, but are notlimited to, are 2-aminoethyl methacrylate, allyl amine, 3-aminostyrene,and the like. After polymerization, amine groups in the polymericcrosslinks may be further crosslinked using difunctional amine reactivecrosslinkers such as, by way of example, and not limitation,glutaraldehyde, EDC, di- or polyepoxides based crosslinkers. Forexample, without limitation, tissue crosslinked using allyl amine isfurther treated with 0.2% glutaraldehyde solution for 24 hours. Theglutaraldehyde treatment creates additional crosslinking tissue proteinsand amine groups of polymerized allyl amine.

In some embodiments, monomers containing phosphorylcholine moiety may beused to crosslink the tissue. Monomers such as 2-methacryloyloxyethylphosphorylcholine (MPC) could be infused into the tissue andhomopolymerized or copolymerized to promote crosslinking and/orgrafting. The MPC polymers and copolymers are well-known for theirexcellent biocompatibility and blood compatibility presumably due tocell membrane like properties of MPC copolymers

In one embodiment, polyvinyl alcohol is incorporated in the tissue. Thisis achieved by polymerizing vinyl acetate monomer in the glycidylmethacrylate modified tissue. The polyvinyl acetate incorporated in thetissue is converted into polyvinyl alcohol by exposing the tissue tomild acid hydrolysis conditions. The polyvinyl alcohol incorporated inthe tissue could be further crosslinked by methods known in the art suchas, by way of example, and not limitation, glutaraldehyde crosslinking,metal ion crosslinking or freezing and thawing. Using a similarapproach, semicrystalline polymer polyacrylonitrile can be incorporatedin the tissue and is then partially hydrolyzed to produce polyacrylicacid. The partially hydrolyzed polyacrylonitrile produces strongmechanical hydrogels.

In another embodiment of the present invention, a monomer which forms apolymer with ability to form complexes with ions, drugs, small moleculesand polymers is used in crosslinking and polymerization. In oneexemplary embodiment, vinyl pyrrolidinone monomer is used to polymerizeand crosslink the modified tissue as (see Example 37 described below).The polymerized polyvinyl pyrrolidinone (PVP) can form complexes withvarious bioactive compounds such as, by way of example, and notlimitation, free iodine, iodide ions and antibiotics. Using a similarscheme, polyacrylic acid introduced by crosslinking with acrylic acidmonomer is complexed with polyethylene glycol. It is understood thatmany other polymer-polymer complexes or polymer-small compound complexescould be used. A monomer-containing cyclodextran may also be used. Thecyclodextran moiety is useful for complexing variety of bioactivecompounds. The selection of such complexes will depend on the propertiesdesired and can readily be practiced by one skilled in the art in lightof the teachings of the present invention. For example, aniodine-polyvinyl pyrrolidinone complex may, for example, and withoutlimitation, be used to impart antibacterial properties to the tissue.Polyacrylic acid or acid groups present in the tissue may be complexedor ionized with silver salts to produce an antimicrobial effect due torelease of silver ions. Many silver salts can be used to form a silversalts; these include, but are not limited to, silver acetate, silverbenzoate, silver chloride, silver carbonate, silver iodide, silveriodate, silver nitrate, silver laureate, silver sulfadiazine, silverpalpitate, silver aspartate, silver succinate, and mixtures thereof.Silver phosphate, silver sulfadiazine, silver citrate, silver lactate,and mixtures thereof are preferred. In one embodiment, the ionizedpolyacrylic acid modified tissue material/collagen implant is incubatedin a concentrated solution of sodium lactate (for example, withoutlimitation, 0.1M to 1.0M) for about 10 to about 60 minutes, mostpreferably about 30 minutes. The materials are then transferred to aselected silver salt solution (e.g., silver lactate) for about 5 toabout 120 seconds, preferably about 60 seconds, in order to produce ananti-microbial surface that retains silver ions and slowly releases themover an extended period.

In another embodiment of the present invention, a di- or polyfunctionalwater soluble macromonomers such as, by way of example, and notlimitation, polyethylene glycol diacrylate or polyethylene glycoldimethaacrylate is used to polymerize and crosslink the animal tissue.In one embodiment, a 5 to 35%, more preferably 10-25%, even morepreferably 23% solution of polyethylene glycol, molecular weight 10000moles/g (PEG10KDA) is used to crosslink the tissue. Briefly, unsaturatedgroups modified tissue is soaked in the PEG10DA solution containingIrgacure 2959 as photoinitiator and heparin as a bioactive compound. ThePEG10DA in the tissue is then polymerized and crosslinked. The tissue iswashed with water to remove unreacted PEG10DA. The heparin entrapped inthe crosslinked tissue-PEG10DA matrix is slowly released. Other monomerssuch as, by way of example, and not limitation, polyethylene glycoldimethaacrylate, polypropylene glycol diacrylate or methacrylate, toname a few, may also be used. Polyethylene glycol diacrylate withmolecular weight from 400 to 100000 Daltons could be used to control themolecular porosity of the crosslinked polymer network. In someembodiments, the monomers containing degradable links are used tocrosslink the tissue. The monomers could be small molecule in nature(typically molecular weight less than 2000) or polymeric macromonomers.In one embodiment, a polyethylene glycol based degradable macromonomeris used. Briefly, a biodegradable monomer is synthesized frompolyethylene glycol. The polyethylene glycol chain is extended usingoligomeric polyhydroxyacid or polylactone links and then terminated withacrylate polymerizable end groups. The polyhydroxyacid or polylactonelinks serve as biodegradable sites in the monomer. These sites undergohydrolysis or biodegradation and break the polymeric crosslinks andmaking the tissue susceptible for enzymatic degradation. In anotherembodiment of the present invention, a liquid hydrophobic biodegradablemonomer is synthesized by polymerization of cyclic lactones andmodifying the terminal group with polymerizable groups such as, by wayof example, and not limitation, acrylic group is used. Briefly,unsaturated group modified tissue and biodegradable monomers arecopolymerized using a free radical polymerization technique such as, byway of example, and not limitation, photopolymerization. Uponcrosslinking, the degradable polyhydroxyacid or polylactone linksundergo hydrolysis and the tissue becomes non-crosslinked and thereforesusceptible for enzymatic degradation. Depending on the type ofpolyhydroxyacid or polylactone used, the crosslinked tissue degradationmay be controlled. The degradation may range from weeks to years. Ingeneral, oligoglycolate polymer crosslinks will degrade in weeks,oligolactate crosslinks will degrade in months and oligocaprolactonecrosslinks will degrade in years. In one embodiment, the hydrophobicliquid biodegradable oligomer is polymerized in the presence of tissuemicroparticles obtained by cryogenic grinding. The polymerization isconducted in a mold and sodium chloride is used as a porosity-inducingcompound. In another embodiment of the present invention, a degradablemonomer containing hydrolizable bond is obtained from hydroxy alcoholssuch as, by way of example, and not limitation, hydroxy amine or ethanolamine. These hydroxy alcohols are reacted with acryloyl chloride ormethacryloyl chloride to form ester-amide derivative. The ester bond issusceptible to hydrolysis upon implantation. Biodegradable monomerscontaining peptide links that can be broken down by enzymes such as, byway of example, and not limitation, collagenease, pepsin, etc. may alsobe used in crosslinking. The tissue-synthetic biodegradable crosslinkedmaterial composite may, for example, and without limitation, be used asscaffold for tissue engineering such as, by way of example, and notlimitation, scaffold for bone tissue engineering. Cells, enzymes orbioactive compounds may be incorporated during polymerization andcrosslinking process to enhance the therapeutic effect.

Di- or polyunsaturated monomers containing degradable bonds have alsobeen disclosed in U.S. Pat. No. 6,713,646 (Zhang, et al) and U.S. Pat.No. 5,410,016 (Hubbell, et al), which are cited here for reference only.Such monomers and their copolymers with other known biocompatiblemonomers can also be used to form biodegradable hydrogels in the tissuematrix. Briefly, monomers described in the above cited references arereacted with tissue modified with unsaturated groups and polymerized asdescribed above. The hydrogels incorporated into the tissue matrixdegrade by hydrolysis making the tissue susceptible to enzymaticdegradation.

In some embodiments, a glutaraldehyde-crosslinked tissue or EDCcrosslinked tissue is first reacted with glycidyl acrylate ormethacrylate to introduce unsaturated groups in the tissue. Theseunsaturated groups are then further crosslinked using various monomersas described above. This approach is especially useful for heart valvebioprosthesis made using bovine pericardial tissue or porcine aorticroot tissue.

In some instances, more than one crosslinking method may be employed toobtain a desirable crosslinked tissue.

In an alternate embodiment of the present invention, the method formaking the tissue is carried out such that part(s) of the tissue arebiostable and/or biodegradable. Table 3 provides a summary ofexperiments performed to stabilize the tissue by various embodimentsdiscussed above.

TABLE 3 Summary of experiments related to biostable tissue stabilized bypolymeric crosslinks. The tissue was first modified with unsaturatedgroups and then crosslinked by free radical photopolymerization usingvarious monomers Visual Observation Unsaturated after 60 day groupMonomer used Shrink Resistance subcutaneous modifying in free radical UVlight Temperature to pepsin implantation Group Type of Tissue reagentpolymerization exposure (° C.) digestion in rat Untreated Bovine Notreatment No Treatment No 60 Digested Substantially Control pericardiumcompletely degraded No BP No treatment Vinyl Yes 60 Digested —Unsaturated pyrrolidinone, completely group control 100% (VP) DarkControl BP Glycidyl 100% VP NO 60 Digested Substantially methacrylatecompletely degraded (GM) VP fixed-1 BP GM 100% VP Yes >95 Very good Nodegradation VP fixed-2 BP GM 100% 2- Yes >95 — — hydroxyethylemethacrylate VP fixed-3 BP GM 20% NIPAM Yes >95 — — VP fixed-4 BP GM 50%NIPAM + Yes >95 — — 50% VP VP fixed-5 BP GM Tetraethyleneglycol Yes >95— — dimethacrylate VP fixed-6 BP GM 2-acrylamido-2- Yes — — —methylpropane sulfonic acid VP fixed-7 Bovine GM 100% VP Yes >95 Verygood No vein degradation VP fixed-8 Bovine GM 100% VP Yes — — — corneaVP fixed-9 Bovine GM 100% VP Yes — — — meniscus VP fixed-10 Sheep skinGM 100% VP Yes >95 Very good — Acrylic acid BP GM 100% Yes >95 Very goodfixed Acrylic acid Acrylamide BP GM 50% acrylamide Yes >95 Very goodfixed in water Acrylamide BP GM 10% acrylamide Yes >95 Very good fixedin water Fumaryl BP Fumaryl 100% VP Yes >95 Very good chloride chlorideMethacrylic BP MA 100% VP Yes >95 Very good anhydride (MA) MethacrylicBP MA 100% VP No. Used 73 — anhydride thermal (MA) initiator

It clear that a variety of tissue types from various animal sources canbe stabilized using the inventive methods described above. In manycases, the tissue did not show shrinkage in boiling water indicatinghigh stability and crosslinking of collagen/protein molecules. Theunmodified tissue, dark control tissue (tissue modified with unsaturatedgroups and infused with monomer and photoinitiator but not exposed tolight) and unsaturated group control (tissue not modified withunsaturated group, but infused with monomer and photoinitiator solutionand exposed to UV light) showed substantial degradation when subjectedto pepsin digestion and low shrink temperature, typically around about60° C. Thermally initiated polymerization also showed higher shrinktemperature. Some fixed and unfixed tissues are subjected to ratsubcutaneous implantation for 60 days. The fixed tissue showed excellentbiostability with no sign of degradation. The untreated tissue and darkcontrol tissue are either completely digested or substantially digestedindicating poor biostability or biodegradation.

Crosslinking of Tissue with Di- or Polymercapto Compounds

The tissue modified with polymerizable unsaturated groups may becrosslinked using di- or polyfunctional mercapto compounds. The modifiedtissue is treated under effective cross-linking conditions with a di- orpolymercapto organic compounds to crosslink the tissue. FIG. 7 is aschematic representation of exemplary steps involved in tissuecrosslinking using mercapto compounds, in accordance with an embodimentof the present invention.

In one embodiment, a method of cross-linking a tissue is provided thattreats the tissue under effective cross-linking conditions with a di- orpolymercapto organic compound.

By “di or polymercapto” is meant any compound including the structure:

SH—R—SH

wherein R is an organic moiety having at least 1 carbon atom and SH is athiol or mercapto group capable reacting with unsaturated carbons suchas, by way of example, and not limitation, acrylamide, methacrylamide,acrylate or methacrylate groups. R may have one or more mercapto groups.

Many di- or polymercapto compounds may, for example, and withoutlimitation, be used in tissue crosslinking. The preferred compoundsinclude, but are not limited to, 1,3-propane thiol, 1,4-butane thiol,1,5-hexane thiol, polyethylene glycol terminated with thiol,trimethylolpropane tris(3-mercaptopropionate), and the like. In onepreferred embodiment, the di- or polymercapto organic compound is atrimethylolpropane tris(3-mercaptopropionate).

Preferably, the di- or polymercapto organic compound is a solute in afluid including a solvent. The fluid including the di- or polymercaptoorganic compound also includes a solvent. The solvent can be any liquidin which the compound is soluble and in which the compound does notundergo degradation or side reactions. If the di- or polymercaptoorganic compound is not soluble in water, but is soluble in organicsolvents, dimethyl sulfoxide (DMSO) or acetonitrile, for example, may,for example, and without limitation, be used as a co-solvent.

The concentration of the di- or polymercapto organic compound in thefluid is preferably between about 0.1 mg/mL and about 100 mg/mL. Morepreferably, the concentration is between about 1 mg/mL and about 20mg/mL.

The pH of the fluid can be any pH which is not deleterious to the tissuebeing treated or the cross-linking reaction. The pH of the fluid can beadjusted by any appropriate technique. Typically, the pH of the fluid isbetween about pH 6 and about pH 10. This pH range allows cross-linkingto be relatively rapid and have a relatively low amount ofside-reactions. Preferably, the pH of the fluid is between about pH 7and about pH 9. More preferably, the pH of the fluid is between about pH8 and about 9.

The temperature of the fluid may be any temperature at which thecross-linking reaction is relatively rapid and a relatively low amountof side reactions occur. Preferably, the temperature of the fluid isbetween about 0° C. and about 45° C. More preferably, the fluidtemperature is between about 0° C. and about 30° C. Conveniently, thereaction may be carried out at room temperature about 25 to about 30° C.

One skilled in the art will readily recognize, in light of the teachingsof the present invention, that the duration of treatment is notcritical, so long as the tissue and the di- or polymercapto organiccompound remain in contact long enough for cross-linking to proceed tothe desired extent. The duration of treatment may vary depending on thetissue being treated or the di- or polymercapto organic compound beingused for cross-linking. Typically, treatment duration is in the range offrom about 1 min to about 24 hr. Preferably, treatment duration is atleast about 30 min, more preferably at least about 6 hr.

Tissue Crosslinking by Hydrogen Abstraction Mechanism

Aromatic ketones such as, by way of example, and not limitation,benzophenone are known to undergo hydrogen abstraction reaction whenexposed to long UV light. In one aspect of the present invention,hydrogen abstraction reaction is exploited for tissue crosslinking. Inone embodiment, a method of crosslinking a tissue is achieved by stepsthat include, without limitation, treating the tissue under effectivecross-linking conditions with a crosslinker capable of undergoinghydrogen abstraction reaction; and crosslinking the tissue by hydrogenabstraction mechanism. Surmodics Inc. has commercialized a technologybased on hydrogen abstraction reaction for medical device coatings andsurface modifications. In one exemplary embodiment (see Example 34described below), a benzophenone derivative of a polymer (polyethyleneglycol terminated with benzophenone) is synthesized. This polymer isthen infused into a tissue and the tissue is exposed to long UV light(360 nm). The benzophenone undergoes hydrogen abstraction reaction andcrosslinks the tissue. Those skilled in the art of surface modificationwill readily recognize, in light of the teachings of the presentinvention, that that many reaction variables can such as, by way ofexample, and not limitation, polymer concentration, exposure time, lightintensity, type of chromophore used may be changed to obtain aneffectively crosslinked tissue. Many polymers that can be modified usingbenzophenone are useful in crosslinking. These include, but are notlimited to, polyvinyl pyrrolidinone, heparin, polyvinyl alcohol,polyethylene glycol and their copolymers, and the like. Many differentchemicals that undergo hydrogen abstraction reaction could be used.Chemicals that undergo hydrogen abstraction reaction in long UV orvisible light range are preferred. One embodiment of the presentinvention provides for a method for forming a crosslinked tissueincluding the steps of providing a tissue suitable for humanimplantation; exposing the tissue to a fluid including a compoundcapable of reacting with a tissue and undergoing hydrogen abstractionreaction when exposed to light; covalently bonding at least portion ofthe tissue functional groups with the a compound capable of undergoinghydrogen abstraction reaction when exposed to light; and crosslinkingthe tissue under effective cross-linking conditions by exposing thetissue to a long UV or visible light.

In addition, proteins in the tissue may be chemically modified using abenzophenone derivative and then crosslinked by exposing to UV light at360 nm. Benzophenone undergoes hydrogen abstraction reaction uponexposure to UV light and undergoes crosslinking. The crosslinkingdensity can be controlled by amount of benzophenone substituted on thetissue proteins. In one embodiment, the present invention provides foran activated derivative of benzophenone carboxylic acid For example,carboxyl groups of 2-carboxyl benzophenone may be activated by formingn-hydroxysuccinimide ester or -hydroxysulfosuccinimide ester and thenreacted with tissue in water (PBS pH 7.2). The benzophenone-modifiedtissue is exposed to high intensity 360 nm UV light to crosslink thetissue.

Compositions and methods for making biodegradable tissue ortissue/synthetic biodegradable polymer composite materials will next bediscussed in some detail.

Tissue Crosslinked Using Degradable Crosslinker

In another aspect of the present invention, a method of cross-linking atissue that is achieved by steps that include, without limitation,treating the tissue under effective cross-linking conditions with acrosslinker containing biodegradable chemical bonds.

A method is herein provided, by way of example, and not limitation, forcross-linking biological tissues to be used in the production ofbiodegradable animal tissue based medical devices and controlled drugdelivery systems. This is achieved by contacting a biological tissue ofinterest with one or more of the disclosed compounds under conditionseffective to cause the desired degree of tissue cross-linking. Thecrosslinks in the crosslinked tissue undergo biodegradation whenimplanted into human or animal body. Upon hydrolysis of the crosslinker,the tissue becomes susceptible to enzymatic degradation and degradesinto non-toxic components. One aspect of the present invention is toprovide a straightforward approach for producing biodegradable animaltissue-based bioprostheses having desirable mechanical andbiocompatibility features and control over degradation of the tissue.

FIG. 8 is a schematic representation of an exemplary tissue crosslinkingusing biodegradable tissue crosslinker, in accordance with an embodimentof the present invention. The biodegradable crosslinkers compoundssuitable for use with this invention can be generally represented by thefollowing structural formula:

F—B—X—F

wherein F is a functional group reactive with collagen, elastin or othertissue-based constituents; and X is an organic molecule or radical atthe core of the crosslinker. X may include Carbon-Carbon,Carbon-Hydrogen, Carbon-Nitrogen, Carbon-Oxygen, Carbon-Sulfur,Nitrogen-Hydrogen and Oxygen-Hydrogen covalent bonds.

F is a functional group that is sufficiently reactive with the collagenmolecules present in the biological tissue to be treated such that thedesired chemical bond is formed. For example, functional groups reactivewith collagen and suitable for use may include, but are not limited to,anhydride, isocyanate, epoxy, n-hydroxysuccinimide, aldehyde or otherprotein reactive functionalities known in the art. It is desirable tohave two or more functional groups per crosslinker for effectivecrosslinking.

B is a biodegradable link between two F moieties. “Biodegradable link”denotes a covalent bond or bonds that will degrade in a biologicalenvironment by either a biologically assisted mechanism, such as, by wayof example, and not limitation, an enzyme catalyzed reaction or by achemical mechanism which can occur in a biological medium, such as, byway of example, and not limitation, hydrolysis.

The biodegradable linkages B may be chosen such that the resultingbiodegradable biocompatible crosslinked will degrade or be absorbed in adesired period of time. Preferably, biodegradable linkages are selectedthat degrade under physiological conditions into non-toxic products. Thebiodegradable linkage may be chemically or enzymatically hydrolyzable orabsorbable. Illustrative chemically hydrolyzable biodegradable linkagesare ester or amide bonds that undergo cleavage under physiologicalconditions such as, by way of example, and not limitation, found inhuman body (pH 7.2). Preferred linkages include, but are not limited to,polymers, copolymers and oligomers of glycolide, dl-lactide, l-lactide,caprolactone, dioxanone, and trimethylene carbonate. Illustrativeenzymatically hydrolyzable biodegradable linkages include peptidiclinkages cleavable by metalloproteinases and collagenease enzymes. Forexample, collagen contains peptide sequences susceptible to degradationby collagenease. Crosslinker containing reactive groups and suchlinkages may be synthesized and used. Additional illustrativebiodegradable linkages include polymers and copolymers of poly(hydroxyacids, poly(orthocarbonate)s, poly(anhydride)s, poly(lactone)s,poly(aminoacid)s, poly(carbonate)s, and poly(phosphonate)s.Biodegradable linkage may also be non-polymeric; these include, but arenot limited to, ester linkages such as, by way of example, and notlimitation, succinate, glutarate, itaconate, and the like.

The biodegradable crosslinker preferably contains at least two Fmoieties and one B moiety per molecule. The number of F groups can begreater than two per molecule and number of B groups can be greater thanone per molecule.

Preferred biodegradable crosslinkers include, but are not limited to,activated acid esters such as, by way of example, and not limitation,n-hydroxysuccinimide esters (NHS esters). Some of the preferred proteincrosslinkers that can synthesized and used as described according topatent WO 9812274, which is cited here for reference only as onesuitable technique. The polyether NHS esters, more preferablypolyethylene glycol glutarate, or succinate esters are most preferred.

The cross-linking agents described can be made using any suitablesynthetic methodologies including, but not limited to, those known toskilled individuals in the art. One preferred approach involves the useof water soluble polyethylene glycol based activated acid ester is used.In one exemplary embodiment (see Example 19 described below), 10 piecesof 1 cm by 1 cm bovine pericardium tissue are transferred to 50 mlpolypropylene centrifuge tube containing 10 ml PBS. 1.0 g 4arm-n-hydroxysuccinimide ester of polyethylene glycolcarboxymethylene-butyric acid, average molecular weight 10000 Daltons(obtained from Shearwater Polymers, 4 arm, product CM-HBA-NS-10K) isadded to the tube and the mixture is vortexed for 5 minutes. The tissueis isolated from the tube, washed with distilled water and lyophilizeduntil further use. The glutarate ester bond in CM-HBA-NS-10K undergoeshydrolysis when exposed to physiological conditions such as, by way ofexample, and not limitation, PBS pH 7.2. After hydrolysis of thecrosslinker, the tissue degrades by normal enzymatic pathways. Tissuecrosslinked with the 4 arm-n-hydroxysuccinimide ester of polyethyleneglycol carboxymethylene-proprionic acid, average molecular weight 10000Daltons has a shorter degradation time as compared to glutarate ester.Thus, by changing the type of ester linkage, degradation of the tissuecan be controlled.

In another illustrative embodiment (see Example 18 described below), anon-polymeric biodegradable crosslinker that is synthesized fromhydroxylamine is used. Briefly, the hydroxy amine is first reacted withsuccinic anhydride. The acid groups formed are then activated by formingn-hydroxysuccinimide ester (NHS ester). The NHS ester is then used tocrosslink the tissue. The hydrolysis of succinate ester makes the tissuedegradable.

The biological tissue of interest is treated with one or more of thecrosslinking agents under conditions effective to cause the desireddegree of tissue cross-linking. The skilled individual will recognize,in light of the teachings of the present invention, that the time oftreatment is not critical as long as the tissue and cross-linking agentremains in contact for a time sufficient to allow the cross-linking tooccur. The time of treatment may vary depending on the type of tissuebeing treated and/or the particular cross-linking agent used. Typically,the length of the reaction will be from about one minute to one day ormore. However, the time of treatment should not be so long as toadversely affect the cross-linked tissue. Thus, cross-linking timesgreater than about one or two days are generally avoided, though suchlengthy times may be appropriate in certain applications. Preferably,the tissue is treated for a period from about one minute to about sixhours, more preferably for about one hour to four hours. The degree ofcross-linking can to some extent be varied by manipulating the time ofthe reaction. A reaction temperature is selected to be effective inpermitting the desired cross-linking reaction to occur while also beingone that does not adversely compromise the integrity of the tissue beingtreated. The identification of an optimal temperature for a particularagent and/or application can be readily determined by the skilledindividual in light of the teachings of the present invention. Thecross-linking reaction can generally be successfully carried out at anambient temperature (20-40° C.), or any other convenient temperatureprovided it does not exceed the tissue denaturation temperature of about60 to about 65° C. Thus, a suitable reaction temperature for use in thisinvention may be from about 0° C. to about 55° C., preferably from about20° C. to about 50° C., more preferably from about 30° C. to about 40°C. This temperature range is generally applicable to all the tissuestreatments described. The tissue is treated under pH conditions that aretissue-stabilizing (i.e., which are not deleterious to the tissue beingtreated) and which do not adversely effect the tissue cross-linkingreaction. This will typically be in a range from about pH 6 to about pH9. More preferably, the pH will be from about 7.0 and about 8.0. Mostpreferably, it will be from about 7.0 to about 7.4. The optimal pH maydepend to some extent on the cross-linking agent employed, the type ofcrosslinker being employed, and/or on the tissue being treated, but canbe readily determined, in light of the teachings of the presentinvention, without undue experimentation by the skilled individual inthis art. The process of cross-linking biological tissues can be carriedout in any suitable solvent. The choice of solvent is generally notcritical; however, preferred solvents will typically be aqueous mediumwith buffering agent. Water-miscible organic solvents that have minimaltoxicity to the tissue and/or the recipient, will be non-denaturing andwill be compatible with the tissue/protein cross-linking reaction. Somesuch solvents include, but are not limited to, linear or branched loweralcohols (i.e., having from about one to four carbons); aprotic highpolarity organic solvents such as, by way of example, and notlimitation, n-methylpyrrolidinone, dimethylsulfoxide, dimethylformamide.dimethylacetamide etc.; lower ketones (i.e., having from about 3 to 6carbons) such as, by way of example, and not limitation, methyl ethylketone or cyclohexanone; and polyhydroxy compounds such as, by way ofexample, and not limitation, glycerol, ethylene glycol, or polyethyleneglycols having molecular weights less than about 1000.

One embodiment of the present invention provides for a biodegradableabsorbable non-crosslinked tissue based implant material includingcovalently bonding at least a portion of the tissue functional groupswith a compound having the formula F—X, wherein F is a functional groupreactive with collagen or protein or components in the tissue;preferably free primary amine groups in the tissue and X is an organicmolecule covalently linking F and X. X may include Carbon-Carbon,Carbon-Hydrogen, Carbon-Nitrogen, Carbon-Oxygen, Carbon-sulfur,Nitrogen-Hydrogen and Oxygen-Hydrogen covalent bonds. X may polymeric ornon-polymeric. For example, and not by way of limitation, the compoundF—X may be selected from polyethylene glycol with tissue reactivegroups; n-hydroxysuccinimide ester of acetic acid, n-hydroxysuccinimideester of acrylic acid, polyethylene glycol monomethoxy alcohols modifiedwith acid group, n-hydroxysuccinimide ester of methacrylic acid; acrylicanhydride; acetic anhydride; n-hydroxysuccinimide ester oftriiodobenzoic acid; succinic anhydride, glutaric anhydride, acetylchloride; acrolein, methacrolein, lactide, glycolide, methyl isocyanate,phenyl isocyanate, glycidyl methacrylate, and glycidyl acrylate. FIG. 9is a schematic representation of an exemplary biodegradableuncrosslinked tissue modified using polyethylene glycol, in accordancewith an embodiment of the present invention. The method shown issuitable for obtaining a biodegradable uncrosslinked tissue modifiedwith polyethylene glycol.

Animal Tissue and Synthetic Biodegradable Polymer Composites

The present invention provides for novel methods and compositions formaterials that combine the properties of animal tissue and synthetic orsemisynthetic biodegradable polymers, copolymer and oligomers. In oneembodiment, the present invention provides for a method for forming ananimal tissue and synthetic biodegradable polymer composite implantincluding the steps of providing a tissue suitable for humanimplantation; exposing the tissue to a fluid including a biodegradablepolymer dissolved in the solvent; and evaporating the solvent. In analternate embodiment, the tissue is exposed to a fluid includingbiodegradable polymer dissolved in the solvent and a therapeutic orbioactive substance dispersed in the solvent. In another alternateembodiment, the present invention provides for a method for forming ananimal tissue and synthetic biodegradable polymer composite includingthe steps of providing a tissue suitable for human implantation;exposing the tissue to a fluid including biodegradable crosslinkablepolymer dispersed in the solvent; evaporating the solvent; andcrosslinking the biodegradable polymer. In these various embodiments,the tissue may be dehydrated. Additionally, the biodegradable polymermay be hydrophilic or hydrophobic.

Animal tissue such as, by way of example, and not limitation, bovinepericardium has unique mechanical properties such as, by way of example,and not limitation, excellent fatigue resistance and durability.However, animal tissues have found little utility in deliveringbioactive compounds, especially water soluble compounds, in a controlledmanner. A composite material including synthetic biodegradable polymerand animal tissue is discussed herein. Methods of such compositepreparation as well as their medical applications are discussed herein.Compositions and methods that physically or chemically bound syntheticbiodegradable polymers to the tissue are also discussed herein.

In one illustrative and preferred embodiment (see Example 22 describedbelow), the animal tissue is dehydrated by exposing it to a series ofaqueous ethanol solutions. The dried tissue is then added to a solutionof synthetic biodegradable polymer in an organic solvent incubated forsufficient amount of time until the polymer solution penetrates thetissue matrix. The polymer solution may include biologically activecompound. The tissue is removed from the solution and the polymersolvent is evaporated or removed by extraction with other solvents. Thetissue-biodegradable polymer composite may, for example, and withoutlimitation, be used in variety of medical and surgical applications. Thetissue may be mechanically or chemically treated to improve adhesion ofbiodegradable polymer to the tissue. Mechanical modification mayinvolve, for example, creating perforations, micro-porosity tomechanically interlock the biodegradable polymer in the tissue matrix.The molecular weight of biodegradable polymer may vary form 1000 to 1million g/mole, most preferably from 2000 to 500000 g/mole and even morepreferably from 5000 to 150000 g/mole. The amount of polymerincorporated in the tissue may range from 1% to 70%, more preferably 5%to 30%.

The animal tissue used may be biostable or biodegradable. The animaltissue used may be crosslinked or non-crosslinked. The animal tissueused may be crosslinked using conventional crosslinking technique suchas, by way of example, and not limitation, EDC crosslinking orglutaraldehyde crosslinking methods known in the heart valvebioprosthesis art. The animal tissue may be non-crosslinked orchemically modified without crosslinking. This includes tissue likeporcine sub-mucosa tissue or non-crosslinked but chemically treatedtissue. Membrane-like biodegradable tissue is most preferred forcontrolled drug delivery applications. In many medical deviceapplications where biodegradable tissue is needed, a non crosslinkedtissue, EDC crosslinked tissue or tissue crosslinked using biodegradablecrosslinker as discussed previously may, for example, be used.Applications were biostable tissue is needed, then glutaraldehyde or dior polyepoxide crosslinked tissue may, for example, be used.

The bioabsorbable or biodegradable polymers used in this inventioninclude any polymer that degrades into non-toxic products upondegradation and may include, but are not limited to, polymers, oligomersor copolymers generally known as polylactones or polyhydroxy acids;polylactones such as, by way of example, and not limitation, polylactidepoly-L-lactide (PLLA), poly-D-lactide (PDLA), poly-DL-Lactide (PLA),polyglycolide (PGA), polylactide-polyglycolide copolymers;polydioxanone, polycaprolactone (PCL), Polyhydroxyalkanoates arepolyesters produced by microorganisms such as poly(3-hydroxybutyrate),3-hydroxyvalerate, 4-hydroxybutarate, 3-hydroxyhexanoate,3-hydroxyoctanoate can also be used. Polycaprolactone-polyglycolidecopolymers, polylactone-polyethylene oxide copolymers are preferred.Modified cellulose, polylactones collagen, poly(hydroxybutyrate),polyanhydride, polyphosphoester, poly(amino acids), poly(alpha-hydroxyacid) or related copolymers materials, each of which have acharacteristic degradation rate in the body, may also be used. Forexample, polyglycolide and polydioxanone copolymers degrade in weeks tomonths. PLA degrades in months to few years and polycaprolactonedegrades in few years. Their copolymers have intermediate degradationtimes depending on the composition of the copolymer. The biodegradablepolymer may be linear, branched, star type. The biodegradable polymermay be block or random copolymer or may be blend of two are morepolymers. The block copolymers include, but are not limited to, ABAtype, BAB type or AB type block copolymers. Bioabsorbable polymers suchas, by way of example, and not limitation, PLLA, PDLA, PGA and othersare commercially available from several sources including PURAC America,Inc.; Aldrich, Polysciences, and Birmingham Polymers. The biodegradablepolymer may be in a variety of forms, for example, liquid, solid, semisolid, wax type or gel type when incorporated in the tissue.

The biodegradable polymer may be covalently linked to the tissue. Manymethods of covalently bonding the polymers may, for example, and withoutlimitation, be used. One preferred and illustrative reaction scheme isshown in FIG. 10. In one preferred and exemplary embodiment (see Example39 or 40 described below), the hydroxy groups of proteins in the tissueor collagen matrix may, for example, and without limitation, be used toinitiate polymerization of cyclic lactones or carbonates. The cycliclactones that may, for example, and without limitation, be used include,but are not limited to, lactide, glycolide, caprolactone, dioxanone,trimethylene carbonate, and the like. Two or more lactones may becopolymerized to obtain a desirable degradation profile of the compositematerial. The polymerization is usually, though not necessarily,catalyzed by metal organic catalysts such as, by way of example, and notlimitation, stannous octoate. The concentration of catalyst added maydepend on the catalyst used. Enzymatic catalysts or biological enzymesthat promote esterification may also be used. Preferred catalyststannous octoate is generally added in the range of about 100 ppm to10000 ppm relative to cyclic lactone concentration. The polymerizationreaction may be carried out in a suitable solvent or under meltconditions to obtain a desired molecular weight of the polylactone. Manycommonly used organic solvents can be used, but solvents that do notdenature the proteins in the tissue are preferred. Preferred commonlyused organic solvent solvents include, but are not limited to, acetone,tetrahydrofuran, benzene, toluene, xylene, chloroform, methylenechloride, dimethyl sulfoxide, dimethylacetamide, and the like. Thepreferred temperature is chosen that promotes rapid polymerization overa period of about 7 days, more typically in about 2 days even moretypically in about a few hours. If using a tissue, polymerizationtemperature below shrink temperature is most preferred. The shrinktemperature of uncrosslinked tissue is generally around about 60° C. andcrosslinked tissue is around about 70-100° C. Polymerization conditionsusing temperatures below about 60° C. are most preferred. Lowertemperature usage generally requires longer polymerization times. Themolar ratio of hydroxy groups in the tissue to lactone may, for example,and without limitation, be used to control the degree of polymerizationof cyclic lactones. For high molecular weight polymers, a higher ratioof cyclic lactone to hydroxy groups in tissue may, for example, andwithout limitation, be used. The molar ratios (cyclic lactone/hydroxygroups) may vary from about 1 to about 500 depending on the finalmolecular weight desired. A molar ratio of about 1 to 50 is generallypreferred. In one embodiment, lactide polymerization was initiated bytissue or solid collagen sponge using THF as a solvent and stannousoctoate as a catalyst. The tissue-polylactide composite has a differentdegradation profile than this tissue. The pepsin digestion data fordl-lactide modified tissue is given in Table 4.

TABLE 4 Pepsin digestion data on dl-lactide modified tissue PepsinVisual Tissue treatment digestion Observations Bovine pericardiumuntreated Completely Complete control degraded dissolution in 6 hours.Bovine pericardium, treated with 45% reduction Partial dl-lactide inpresence of stannous in 51 hours digestion octoate after 51 hours Bovinepericardium crosslinked No degradation No change with glutaraldehyde,treated with in tissue dl-lactide in presence of stannous appearance.octoate

The data in Table 4 indicates that the uncrosslinked tissue treated withdl-lactide showed substantial stability towards enzymatic degradation ascompared to untreated tissue which degraded in 6 hours. Howeverdl-lactide treated tissue lost 45% weight in 51 hours indicating itssusceptibility to enzymatic degradation. Thus, it is expected that thedl-lactide tissue can resist degradation much longer than the untreatedtissue and thus stay much longer than untreated tissue once implanted inthe body. As expected, glutaraldehyde treated tissue shows higherstability towards enzymatic degradation. Collagen based films, fibermats, sponge, porous scaffolds may also be modified using cycliclactones.

In another embodiment, dl-lactide polymerization was initiated bygelatin or collagen to produce a gelatin-polylactide graft copolymer.The composite degrades by hydrolysis and an enzymatic degradationmechanism. The degradation of synthetic polymer will depend on cycliclactone used. In general, polyglycolide degrades in about a few months,polydl-lactide in about 6 to 18 months, and polycaprolactone in about afew years. Their copolymers may, for example, and without limitation, beused to achieve intermediate degradation times. The synthetic polymermay, for example, and without limitation, be used to entrap a bioactivecompound inside the biodegradable polymers. Techniques such as, by wayof example, and not limitation, solvent diffusion may, for example, andwithout limitation, be used to diffuse or entrap drugs in the degradablepolymer matrix in the composite material.

The biodegradable polymers may be hydrophobic or hydrophilic. Thebiodegradable polymers may be crosslinked or non-crosslinked. Thehydrophobic polymers include, but are not limited to, polymers,copolymers or oligomers of glycolide, dl-lacide, d-lactide, l-lactide,caprolactone, dioxanone and trimethylene carbonate; polyhydroxyacids,polylactic acid, polyglycolic acid, polyanhydrides, and polylactones.Hydrophobic polymers also include polyhydroxyalkanoates which arepolyesters produced by microorganisms such as poly(3-hydroxybutyrate),3-hydroxyvalerate, 4-hydroxybutarate, 3-hydroxyhexanoate,3-hydroxyoctanoate. Preferred hydrophilic polymers include, but are notlimited to, polyethylene glycol-polyhydroxy acid or polyethyleneglycol-polylactone copolymers (PEG-PL copolymers), polyvinyl alcoholco-polylactone copolymers, and derivatives of cellulose, hyaluronic acidand dextran. The PEG-PL copolymers are most preferred. PEG-PL copolymerssuch polyethylene glycol-polylactone copolymers can have a range ofproperties from hydrophobic to hydrophilic depending on the amount ofPEG incorporation in the copolymer and molecular weight of PEG andpolylactone. PEG incorporation that gives up to about 5-70 percent waterabsorption is preferred. The PEG-PL copolymers with PEG molecular weightabout 400 to about 30000 Daltons are preferred. In one exemplaryembodiment (see Example 21 described below), a PEG molecular weight 8000(Carbowax 8000) is used in the polymerization of dl-lactide usingstannous octoate as a catalyst. The polymerization reaction is carriedout for 16 h at 160° C. The polymer is isolated and incorporated in thetissue (PEG8K50). Briefly, a membrane-like tissue such as, by way ofexample, and not limitation, pericardial tissue is incubated for 30minutes each in 20 percent ethanol, 40 percent ethanol, 80 percentethanol, and finally in 100 percent ethanol to dehydrate the tissue. Thetissue may also be dehydrated by lyophilization. The dehydrated tissueis then transferred into polyethylene glycol-lactate copolymer (PEG8K50)and rifampin solution in chloroform and incubated for 2 hours. Thetissue is removed from the solution and the solvent is evaporated by airdrying. The reddish yellow rifampin-PEG-lactate polymer is clearlyvisible to the naked eye. The dry tissue is sterilized using ethyleneoxide and used as a biodegradable drug delivery patch. The polyethyleneglycol-lactate polymer in the tissue acts as a hydrophilic biodegradablepolymeric drug delivery matrix for Rifampin.

The biodegradable polymer incorporated into the tissue may becrosslinked in nature. The crosslinked polymer may be hydrophilic orhydrophobic. It is preferred that the crosslinking reaction is carriedout after infusion of precursor polymers in the tissue. This permitsbetter distribution of polymer in the tissue matrix. In one exemplaryembodiment (see Example 15 described below), a hydrophobic freeradically polymerizable liquid precursor is made by initiating thepolymerization of lactide using trimethylol propane triol and thenendcapping the terminal hydroxyl groups with acrylate end groups. Thispolymerizable precursor is then infused in the tissue along withphotoinitiator and vinyl pyrrolidinone as comonomer using chloroform asa solvent. After the solvent removal, the lactide precursor isphotopolymerized exposing the tissue to long wavelength light. Inanother variation (see Example 20 described below), a water-solubleprecursor based on polyethylene glycol is incorporated into the tissuefirst and then crosslinked. If a bioactive compound such as, but notlimited to, heparin, rifampin, or growth factor is mixed prior tocrosslinking, then the compound remains entrapped in the crosslinkedpolymer and is then released slowly upon degradation of biodegradablecrosslinked polymer.

Radio-opaque implantable tissueAnimal tissue such as, by way of example,and not limitation, porcine pericardium or bovine pericardium is poorlyvisible when viewed using standard medical x-ray imaging techniques.This is probably due to poor absorption of x-rays by the components ofthe tissue. In many medical device applications, especially in surgicalprocedures which are done using x-ray fluoroscopic techniques; it isdesirable to have an implantable tissue that can be differentiated fromsurrounding tissue when implanted and viewed under standard medicalx-ray imaging technique. A radio-opaque tissue can be easily seen whenviewed using standard medical x-ray imaging technique. This inventionprovides compositions and methods for making radio-opaque implantabletissue. Some of the preferred methods for making implantableradio-opaque tissue are given below.

Implantable animal tissue may be chemically modified using a reagentincluding at least one functional group capable of reacting with tissue(F) and at least one radio-opaque chromophore capable of absorbingx-rays (M). The chemical modification agents used in accordance with anembodiment of the present invention include an organic functionalmolecule with at least one functional group capable of reacting withtissue and at least one radio-opaque chromophore. Thus, the compoundssuitable for use with this invention can be generally represented by thefollowing structural formula:

F—X-M

wherein F is a functional group reactive with collagen or protein orcomponents in the tissue; preferably free primary amine groups in thetissue; X is an organic molecule/radical covalently linking F and X. Xmay include Carbon-Carbon, Carbon-Hydrogen, Carbon-Nitrogen,Carbon-Oxygen, Carbon-Sulfur, Nitrogen-Hydrogen and Oxygen-Hydrogencovalent bonds. X may polymeric or non-polymeric.

M is a chromophore that can absorb x-rays. The preferred chromophoresinclude, but are not limited to, phenyl ring compounds such as, by wayof example, and not limitation, 2,3,5-triiodobenzoic acid,3,4,5-triiodophenol, erythrosine, rose bengal,3,5-Bis(acetylamino)-2,4,6-triiodobenzoic acid,3,5-Diacetamido-2,4,6-triiodobenzoic acid, heavy metal ion complexes,and the like. The iodine in these compound may be radioactive ifdesired. A radioactive (I¹²⁵ or I¹³¹) could be potentially used forlocal radiation therapy.

F is a functional group that is sufficiently reactive with the collagenmolecules present in the biological tissue to be treated such that thedesired chemical bond is formed. For example, functional groups reactivewith collagen and suitable for use may include, but are not limited to,anhydride, isocyanate, n-hydroxysuccinimide, n-hydroxysulfosuccinimide,epoxy, aldehyde or other collagen reactive functionalities known in theart. It is desirable to have only one F moiety per molecule to avoidcrosslinking of proteins in the tissue. If crosslinking is desired, morethan 1 F moiety may, for example, and without limitation, be used.

Preferred molecules which are capable of modifying the tissue include,but are not limited to, activated monofunctional triiodobenzenederivatives such as, by way of example, and not limitation,n-hydroxysuccinimide esters or n-hydroxysulfosuccinimide esters oftriiodobenzoic acid or erythrosin.

The tissue modification using reagents, which are taught by way ofexample in the present detailed description, can be made using anysuitable synthetic methodologies, including but not limited to, thoseknown to skilled individuals in the art. In one embodiment of thepresent invention a method of modifying the issue is achieved by stepsthat include, without limitation, treating the tissue under effectivemodification conditions with a monofunctional tissue reactive iodinatedorganic compound. In one exemplary embodiment (see Example 36 describedbelow), bovine pericardium tissue is modified with triiodobenzoic acidsuccinimide ester (TIBA-NHS). The modification method includes exposingthe tissue to a fluid including reactive iodinated compound that canreact with amino groups in the tissue under mild conditions. As usedherein, the term “activated” as applied to an acid moiety-containingcompound containing an additional moiety such that the activated acidcan react with amino groups under mild conditions. Preferred activatingmoieties include, but are not limited to, disuccinimidyl moieties,n-hydroxy disuccinimidyl moieties, sulfo-disuccinimidyl moieties, andmixtures thereof. Briefly, bovine pericardium pieces, cut from a freshlyobtained bovine pericardial sac, are treated with TIBA-NHS dissolved indimethyl sulfoxide or PBS buffer. The reaction is carried out in aqueousmedium buffered with PBS (pH 7.2). The modification reaction is carriedout for 6 hours at ambient temperature (25° C.) and then for 12 hours at4° C. with gentle shaking. Some NHS esters such as, by way of example,and not limitation, acetic acid NHS esters are sparingly soluble inwater therefore a solubility enhancing compound such as, by way ofexample, and not limitation, dimethyl sulfoxide may, for example, andwithout limitation, be used to facilitate the reaction between activatedacid groups and primary amine groups on the tissue. The reaction occursin mild conditions, in water (PBS and at pH 7.2) and is usually completein about 24 hours, more preferably within about six hours. It can alsobe substantially completed in about 10 minutes and therefore may permitthe use of this method in the operating room during a surgical procedureto modify an autologous tissue. In another variation of this method, themodification reaction may be carried out under mild acidic conditions(IVIES buffer, pH 6.5). In this case, the NHS ester is added every 1.5hours to replace the hydrolyzed NHS ester. (NHS ester is deactivated byreaction with water). The NHS groups react with primary amines groups ofthe tissue forming stable amide bonds. The NHS ester reacts with primaryamines groups on the tissue. Reaction variables such as, by way ofexample, and not limitation, time, temperature, concentration, pressuremay be controlled in such a way that about 1 to about 100 percentprimary amine groups on the tissue are modified. More preferably, about10 to about 95 percent amine groups are modified, even more preferablyabout 40 to about 95 percent amine groups are modified.

In another approach, triiodobenzoic acid is reacted with tissue using1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) as acatalyst. EDC promotes the reaction between carboxylic acid oftriiodobenzoic acid and OH and primary amine groups present in thetissue. EDC treatment also results into crosslinked non-cytotoxictissue. Aqueous medium, more specifically buffered medium, with pH about5.5 to about 7.5 may, for example, and without limitation, be used forsuch reactions. A co-catalyst such as, by way of example, and notlimitation, n-hydroxysuccinimide or n-hydroxysulfosuccinimide may alsobe used to accelerate the coupling reaction. Using a similar approach,other biocompatible hydrogels such as, by way of example, and notlimitation, hyaluronic acid, chitosan and alginate may also be maderadio-opaque and are considered part of the present invention.

In another approach, a radio-opaque polymerizable monomer may, forexample, and without limitation, be used in polymerization andcrosslinking as discussed above. For example, a polymerizableradio-opaque monomer may be obtained by esterification of triiodobenzoicacid and 2-hydroxyethyl methacrylate. The radio-opaque ester may becopolymerized and crosslinked with unsaturated group modified tissue.

In another approach, heavy metal biocompatible salts such as, by way ofexample, and not limitation, silver salts may be deposited in the tissueto make it radio-opaque. Many methods for silver salt incorporation canbe used. In one exemplary embodiment, a tissue is first exposed tosilver nitrate solution by dipping the tissue and removing the tissueand then exposing to sodium acetate or sodium iodide or sodium chloridesolution. The exposure to acetate or chloride ions form insoluble saltsinside the extracellular matrix of the tissue and leach out over aperiod of time. The silver salts provide visibility in x-ray basedmedical imaging equipment.

Tissue used in modification may be crosslinked or non-crosslinked. Anon-crosslinked tissue generally yields biodegradable implantabletissue. A glutaraldehyde crosslinked tissue gives biostable tissue.

The tissue also may be coated with polymeric compositions includingradio-opaque compounds. For example, biodegradable polymers such as, byway of example, and not limitation, poly(lactic acid) and x-ray contrastagent such as, by way of example, and not limitation, iohexyl aredissolved in common organic solvent such as, by way of example, and notlimitation, dimethyl sulfoxide or tetrahydrofuran and the solution maybe spray-coated on the tissue. Upon solvent removal, iohexyl remainsentrapped in the polymeric coating which makes the tissue radio-opaque.

The radio-opaque tissue may, for example, and without limitation, beused to fabricate many and varied medical devices. The medical devicesmay be existing medical devices such as, by way of example, and notlimitation, a heart valve or a vascular graft or devices that are yet tobe developed. More specifically, radio-opaque tissue may, for example,and without limitation, be used to fabricate tissue covered coronary orvascular stents or stent grafts. The visibility of radio-opaque tissuepermits localized drug or device therapy using standard techniques usedin MIS surgery. This radio-opaque tissue may be subjected to additionaltreatments such as, by way of example, and not limitation,anti-calcification treatments known in the prior art.

In some applications, modified non-crosslinked radio-opaque tissue isexpected to undergo degradation after in vivo implantation. Themodification compounds and their degradation products/components must beable to be eliminated from the host body. The modification compounds maybe chosen such that the resulting biodegradable biocompatiblecrosslinked tissue will degrade or be absorbed in a desired period oftime. Preferably, modification reagents are selected in a manner that itwill degrade under physiological conditions into non-toxic products.

In applications where biodegradable tissue is desired, the tissue may beheat-treated to make it denatured. Such denaturing is done to acceleratethe degradation of the tissue. The denaturing may be done by heating amembrane-like tissue such as, by way of example, and not limitation,porcine pericardium in saline solution and holding it in the temperaturerange of about 70 to about 100° C. (above shrink temperature) for about1 minute to about 24 hours, more preferably from about 1 minute to about30 minutes.

Coated Bioprosthetic Tissue Compositions and Methods

Properties of biological tissue surface can be changed by applyingdifferent types coatings. For example, the glutaraldehyde fixed tissuesurface is cytotoxic and does not support cell growth. A surface coatingthat eliminates the surface toxicity is highly desirable.Non-crosslinked tissues such as, by way of example, and not limitation,pericardial tissue supports cell growth and degrade by enzymatic processwhen implanted in the human or animal body. In some applications, suchas, by way of example, and not limitation, prevention of post operativeadhesions or hernia repair application, a barrier surface that ishydrophilic, biocompatible but does not support cell growth isdesirable. A polyethylene glycol based hydrogel on tissue surface mayprovide such non-cell adhesive surface.

In one aspect of the present invention, various coating compositionsthat can be applied on the implantable tissue surface are provided. Thecoating may cover all surface areas of the tissue or may be applied onsome parts of the tissue. FIG. 11 is a schematic representation of anexemplary membrane-like tissue modifications (A-F), in accordance withan embodiment of the present invention. Illustrated in the Figure arevarious modifications of membrane-like implantable tissue. FIGS. 11 Aand B illustrate the coating of a membrane-like tissue such as, by wayof example, and not limitation, bovine pericardium from one and bothsides. The coating may include, for example, cells and/or bioactivecompounds. The coating may also be a biodegradable biocompatiblehydrogel. Multiple layers may also be utilized. Methods for forming suchcoated tissue surfaces and their biomedical applications such as, by wayof example, and not limitation, barrier for post operative adhesionprevention and controlled drug delivery are also described.

In some embodiments, the tissue is coated using a biodegradable polymer.The coated tissue structure is schematically shown by way of example inFIG. 11. The coating thickness may be about 0.1 microns to about 5 mmthick. More preferably the coating may be about 5 micron to about 2 mmthick. The biodegradable coating may degrade in approximately days toyears. The degradation time will depend on the type of biodegradablepolymer used in the coating. For example, a coating based onpolyglycolide polymers and copolymers may degrade in weeks to months,PLA or PLA-PGA coating will degrade in months to years, andpolycaprolactone based coating will degrade in years. An elastomericcoating formed from biodegradable polyurethanes may also be used. Thecoating may have biologically active compounds encapsulated in thepolymer. The coatings also may have cells such as, by way of example,and not limitation, stem cells incorporated in the coating. Thehydrophilic coatings include biodegradable hydrogel coatings formed bycrosslinking and polymerization of polyethylene glycol based watersoluble macromonomers. In one such illustrative embodiment (see Example23 described below), a polyethylene glycol based macromer is firstsynthesized. An aqueous solution of macromer is then polymerized on thetissue to form a thin hydrogel coating.

In another embodiment of the present invention, a syntheticbiodegradable hydrogel is coating is formed by condensationpolymerization (see Example 24 described below). Briefly, the hydrogelis formed by mixing reactive precursor species including nucleophilicfunctional groups and reactive precursor species including electrophilicfunctional groups. In one exemplary embodiment, solutions ofpolyethylene glycol NHS derivatives and dilysine are sprayed andpolymerized on the 10 cm×5 cm bovine pericardial sac. The solutions ofpolyethylene glycol NHS derivatives and dilysine are commercially soldunder the trade name of DuraSeal™ by Confluent Surgical, Waltham, Mass.DuraSeal™ sealant can also be used to make a coated tissue. TheDuraSeal™ coating may be applied just prior to implantation.Alternatively tissue such pericardial patch tissue and DuraSeal™ productand spray systems may be supplied as a kit to make a coated tissueproduct in the surgical suit. The tissue used may be crosslinked(biodegradable) or crosslinked (biostable). For example, AlloDerm® aprocessed human skin tissue marketed by LifeCell corporation may becoated using DuraSeal™ and used. In another embodiment of the presentinvention, a tissue is first dehydrated by lyophilization or using othertechniques known in the art. The precursors may be applied as a drypowder without crosslinking. Upon application on the surgical site, theprecursor powder coated tissue undergoes hydration and crosslinking.

In another embodiment of the present invention, a tissue is firstdehydrated by lyophilization or using other techniques known in the art.A biodegradable polymer dissolved in organic solvent is sprayed on thetissue surface. Alternatively the polymer may be coated using a dipcoating method. The solvent is removed and the polymer coated tissue isrecovered and stored appropriately. The coating may be applied on oneside or on both sides.

A non-crosslinked tissue such as, by way of example, and not limitation,bovine pericardial tissue typically degrades in 4 months when implantedsubcutaneously in the rat body. Control over tissue degradation time ishighly desirable. A polymeric biodegradable coating on the tissue maytemporarily restrict the access of degradative enzymes when implanted inthe body. After the coating is resorbed due to biodegradation processesin the body, the access of enzymatic degradative sites in the tissue isrestored and the tissue is degraded. Thus, coatings may help to lengthenthe degradation time of the non-crosslinked biodegradable tissue. Bychoosing a biodegradable polymer that degrades from months to years,biodegradation of the tissue may be varied from months to years. Thepolymer coating may be loaded with drug for a local or systemictherapeutic effect.

The coating in the degradable polymer-coated tissue, which are taught byway of example in the present detailed description, may be supplementedwith collagenease, pepsin or other protease enzymes which can acceleratetissue degradation. The enzyme is released from the coating in acontrolled manner which then degrades the tissue. This approach may, forexample, and without limitation, be used to accelerate the tissuedegradation.

The tissue-synthetic biodegradable polymer composite material or coatedproduct produced according to the present invention can be modifiedfurther, if necessary or desired, by the addition of anypharmaceutically acceptable compound or agent such as, but not limitedto, antioxidant, plasticizer, coloring agent, x-ray or MRI imagingagent, filler such as, by way of example, and not limitation, calciumphosphate salts and the like.

Another aspect of the present invention is that the tissue basedmaterials described can be used to deliver biologically activecompounds. The composite/coated material will deliver a therapeuticallyeffective amount of bioactive substance in a controlled manner. In oneembodiment, rifampin, a commonly used antibiotic is incorporated in thetissue-biodegradable polymer composite material. Small organic andinorganic molecules as well as large molecules such as, by way ofexample, and not limitation, proteins, carbohydrates, organic molecules,synthetic peptides, genes, antibodies, enzymes, etc. can be used. Someexamples of bioactive compounds that can be released inside the human oranimal body include, but are not limited to, antiviral agents;antiinfectives such as, by way of example, and not limitation,antibiotics; antipruritics; antipsychotics; cholesterol or lipidreducing agents, cell cycle inhibitors, anticancer agents,antiparkinsonism drugs, HMG-CoA inhibitors, antirestenosis agents,antiinflammatory agents; antiasthmatic agents; antihelmintics;immunosuppressives; muscle relaxants; antidiuretic agents; vasodilatorssuch as, by way of example, and not limitation, nitric oxide or nitricoxide adducts; beta-blockers; hormones; antidepressants; decongestants;calcium channel blockers; growth factors such as, by way of example, andnot limitation, bone growth factors, bone marrow proteins, vascularendothelial growth factor, platelet derived growth factor, acidic growthfactors, basic growth factors, wound healing agents, analgesics andanalgesic combinations; local anesthetics agents, antihistamines;sedatives; angiogenesis promoting agents; angiogenesis inhibitingagents; tranquilizers, and the like. In many instances, the duration ofthe drug release is affected by the hydrophobicity of the drug andpolymer, drug loading and polymer composition and polymer degradationcharacteristics. The amount of bioactive compound incorporated in thetissue composite materials is in the range of about 0.1 percent to about90 percent, more preferably in the range of about 5 to about 70 percent,and even more preferably in the range of about 10 to about 50 percent.In some cases, two or more bioactive compounds may, for example, andwithout limitation, be used to achieve a desirable therapeutic effect.

In a biodegradable drug delivery patch application, membrane-liketissues such as, by way of example, and not limitation, porcinepericardial tissue, processed human skin tissue (AlloDerm®), bovinepericardial tissue or porcine sub-mucosa tissue are preferred. Theanimal tissue used may be crosslinked or non-crosslinked. Anon-crosslinked tissue may, for example, and without limitation, be usedif an absorbable patch material is desired. A drug delivery patch couldbe formulated to incorporate a bioactive compound. The patch willdeliver a therapeutically effective amount of bioactive substance in acontrolled manner. The patch may be coated with biodegradable hydrogelsencapsulated with cells or bioactive growth factors. The patch can beutilized to deliver a drug or bioactive compound systemically orlocally. In one embodiment, the present invention provides for a methodfor localized drug delivery including the steps of providing a steriletissue suitable for human implantation; providing a sterilebiodegradable polymer; providing a sterile solvent for biodegradablepolymer; dissolving the biodegradable in the solvent and optionallyadding bioactive compound; exposing the tissue to a polymer solution;forming a coating of polymer on the tissue; and implanting the tissue.

This invention also anticipates the use of non-polymeric liquid/solidsas coating materials for the tissue and as controlled drug deliverymatrices incorporated into the tissue. The non-polymeric materials usedto coat the tissue or to form a composite material include biocompatiblenon-polymeric liquids and solids which can be implanted in the humanbody. These non-polymeric materials include those that have a history ofhuman use. These include, but are not limited to, wax materials such as,by way of example, and not limitation, bone wax, fatty acids and theirderivatives such as, by way of example, and not limitation, stearic acidand oleic acid, sugar derivative such as, by way of example, and notlimitation, sucrose acetate, mineral oil, peanut oil, cotton seed oil,tocopherol, polyethylene glycol, and the like. PEG-Polylactone basedcopolymers with melting below 60° C. are most preferred in thisapplication.

Animal tissue, preferentially a biodegradable tissue, can be used ascarrier for biologically active substances such as, by way of example,and not limitation, sparingly water-soluble drugs without the use ofcarrier. Some examples of such drugs include, but are not limited to,chlorhexidene acetate, chlorhexidene gluconate, lovastatin, simvastatin,paclitaxel, silver iodide, silver acetate, silver lactate, and the like.In one embodiment, chlorhexidene acetate or chlorhexidene gluconate isincorporated in the tissue using a solvent method. Briefly dehydratedtissue is exposed to an organic solvent or semi aqueous solvent such aswater alcohol mixture in which the drug is dissolved. The solvent isremoved and crystals of drug are formed inside the tissue matrix. Thesecrystals serve as reservoir for the drug to be released. The drug isreleased due to slow dissolution of drug crystals. In anotherembodiment, poorly water soluble silver salts such as, by way ofexample, and not limitation, silver chloride, silver iodide, silveracetate, silver lactate and the like are precipitated inside the tissueor extracellular matrix by a reaction of two solutions. Afterprecipitation, sparingly soluble silver salts release silver ions fromthe tissue matrix providing antimicrobial activity to the tissue.Depending on the solubility, different silver salts such as, by way ofexample, and not limitation, silver iodide, silver acetate, silverlactate and the like may be chosen to have different silver ion releaseprofiles. Silver salts can be deposited using many methods such directsalt deposition, ion beam implantation and the like. In a preferredembodiment, silver salts are formed in situ inside the tissue. In onepreferred embodiment, tissue is first exposed to silver nitrate solutionby incubation in silver nitrate solution followed by incubation in asolution of sodium acetate. The silver nitrate in the tissue isconverted into silver acetate in situ which precipitates in the tissuematrix. The concentration of silver nitrate and silver acetate solutionwill very depending on the amount of silver salt is needed in thetissue. In the preferred embodiment, silver ion leaching of 10-100microgram of silver per cm square of tissue surface is preferred. Thedeposited silver crystals may also improve radio-opacity of the tissue.

In many applications, tissue prepared according to an embodiment of thepresent invention may be cut into several geometric shapes. These shapesare then used to construct unique medical devices such as, by way ofexample, and not limitation, heart valve bioprosthesis, vascular grafts,tissue covered stents, and stent grafts. For example, a bovinepericardial tissue may be cut and sewn to form a heart valvebioprosthesis. In some applications, a perforated tissue may, forexample, and without limitation, be used to form a composite material.Also in some applications, the tissue surface may be textured usingdifferent shapes, holes, grooves to promote healing or to improveadhesion of biodegradable polymer in the tissue or to deposit controlleddrug delivery compositions. In some applications, multiple coatinglayers of tissue may, for example, and without limitation, be used. Forexample, a first coating layer may consist of drug and polymer and asecond coating layer on top of first layer may consist of only polymerwithout a drug. Such a coating may act as a diffusion barrier to controlthe release rate of the drug.

Embodiments of the present invention also include the use of grooves orother geometric shapes created on the tissue surface and their use tofill the biodegradable controlled drug delivery compositions. FIG. 11illustrates this concept by way of example, in particular FIGS. 8C and8D. Grooves may be of any shape and pattern. The groove depth and sizemay depend on the tissue thickness and intended use. Generally groovedepth may range from 1 to 2000 micron most preferably from 20 to 1000micron. Many groove shapes may be used these include but not limited towedged shape, rectangular shape, cylindrical shape and the like. In amembrane-like tissue such as, by way of example, and not limitation,porcine pericardium or bovine pericardium, 10 micron to 2 mm diameterholes (i.e., cylindrical grooves) are created using any known method inthe art such as, by way of example, and not limitation, laser drilling,mechanical drilling and the like. The holes or grooves are then filledwith the liquid or solid drug delivery compositions, e.g., controlledrelease polymer compositions containing bioactive compounds. Forexample, the holes are filled with bioerodable or biodegradable polymerwith bioactive compound such as, by way of example, and not limitation,Lovastatin or Atrovastatin (HMG-CoA inhibitor). The bioactive compoundis then released from the polymeric matrix. FIGS. 8E and 8F illustrate,by way of example, and not limitation, the use of perforated tissue.This tissue may be coated with biodegradable polymer with bioactivecompound. The membrane tissue used may be biostable (crosslinked) orbiodegradable (non-crosslinked).

A composite tissue-biodegradable composite material or coated tissuematerial according to an embodiment of the present invention can beformulated so that they can be delivered using minimally invasivesurgical (MIS) techniques such as, by way of example, and notlimitation, laparoscopy, thoroscopy and the like and thus are useful forlocalized drug delivery inside a living body. One embodiment of thepresent invention provides for a method for the treatment of a medicalcondition at a localized site, including the steps providing aimplantable membrane like animal tissue including a bioactive compound;compacting the tissue to a size suitable for implantation usingminimally invasive technique; transporting the compacted tissue to alocalized treatment site; uncompacting the tissue to its original size;and immobilizing the tissue on the treatment site wherein theimplantable tissue and a bioactive compound assists in the treatment ofthe medical condition. In one exemplary approach, the pericardialtissue-polymer composite patch loaded with rifampin is rolled into tubeor other compacted shape so that it can be transported to a localizedsurgical site using a minimally invasive surgical (MIS) technique. Thedrug delivery patch material is then unrolled to form a flat membrane atthe surgical site. The patch is then applied on the tissue surface andconformed to the localized tissue or organ geometry. The patch issecured in place by suturing or stapling to the surrounding tissue. Thesuture or staple may be biodegradable or biostable. The patch is thenused to releases the drug in the localized surgical environment

In some embodiments, a composite of fixed tissue such as, by way ofexample, and not limitation, discussed in this invention orglutaraldehyde fixed tissue is used to incorporate high molecular weightbiocompatible biostable polymers such as, by way of example, and notlimitation, Teflon® (polytetrafluoroethylene) or polyethylene vinylacetate copolymers or their derivatives or polysulfones may beincorporated using a solvent technique. In one example, amorphousTeflon® is dissolved in a proprietary halogenated solvent obtained from3M Corporation. A dehydrated glutaraldehyde fixed bovine pericardialpatch tissue is then exposed to the Teflon® solution. The solvent isthen evaporated. The Teflon® polymer-fixed tissue composite is used as abiostable composite patch for variety of surgical applications.Alternatively expanded, partially expanded or unexpandedpolytetrafluoroethylene (PTFE) membrane may be used to make a tissuebased composite patch. The PTFE membrane may be sewn to the using PTFEbased sutures. The composite patch may be useful as hernia surgeryapplication.

Use of Tissue Engineered Material for Making Bioprosthesis

This invention also teaches of compositions and methods wherein thetissue used in making bioprosthesis is obtained from non-animal sourcelike cell culturing or tissue engineering technologies. The use ofanimal tissue in bioprosthesis manufacture is known for quite some time.However, animal tissue use has certain limitations such as size of themaximum single piece that can be derived from the animal. Alsochemical/protein composition of the animal skin tissue used inconventional animal cannot be changed and is dependent on the speciesand type of tissue used. The tissue derived from animal tissue may alsocontain pathogenic viruses and proteins such as Bovine SpongiformEncephalopathy (BSE). The use of membrane like tissue, made using tissueengineering technologies, overcomes these limitations. In thisinvention, membrane like tissue that may be useful to makebioprostheses, is derived from modern tissue engineering technologieswhich are known in the tissue engineering art or yet to be developed. Inone exemplary embodiment, a sterile porous scaffold is seeded withsmooth muscle cells along with tissue culture medium that supports thegrowth of such cells. The scaffold, cells and cell culture media areincubated at 37° C. in an atmosphere that supports mammalian cell growth(controlled carbon dioxide, humidity etc). Care is taken to minimizebacterial or fungal contamination of the scaffold or culturing cells.The incubation of cells is continued until cells attach, grow and createtheir own extracellular matrix tissue. Additional cell seeding, cellculture media exchanges may be done to achieve a desired tissuethickness is reached. The preferred thickness of engineered tissue mayrange form 1 micron to 3000 micron, most preferably 50 microns to 1500microns. The preferred size of engineered tissue is >1 square inch, morepreferably 1 to 1000 square inch, even preferably 1 to 150 square inch.

The biodegradable scaffold materials that can be used to make theengineered tissue include but not limited to are: degradable polyesterssuch as polyhydroxy acid, polylactones, polyhydroxyalkanoates which arethe polyesters produced by microorganisms such aspoly(3-hydroxybutyrate), 3-hydroxyvalerate, 4-hydroxybutarate,3-hydroxyhexanoate, 3-hydroxyoctanoate. Other degradable polymersinclude but not limited to are: polyamides, collagen, gelatin,polyanhydrides, polyglycolate, polyglycolic acid, polycaprolactone,polycarbonates such as polytrimethylene carbonate, degradablepolyurathanes, fibrinogen, and the like. Copolymers or blends of suchmaterials may also be used. The biodegradable scaffold materials may bethe form a fiber such as polyester fiber, collagen fiber, silk fibers,polyamide or nylon fibers which may be woven to produce a suitablescaffold. The scaffold used manufacturing of engineered tissue may beporous or non-porous. Many methods of introducing porosity are known inthe art. These methods include but not limited to: leaching of solublesalts from the polymer matrix, lyophilization of hydrogel materials,sublimation of solvents or porosity generating compounds from thepolymers and the like. The porosity is chosen to promote rapid cellattachment and growth. The porosity of scaffold may range from 1 micronsto 1000 microns, most preferably 10 microns to 500 microns to promotecell infiltration and growth. The scaffold surface may be chemically orphysically modified using bioactive compounds or peptides that promotecell attachments. Such compounds include but not limited to peptidessuch as RGD, RGDS, IGD and the like. The size, shape and geometry ofscaffold may vary depending on the intended use. A membrane likescaffold with size ranging from 1 square inch to 1000 square inch may beused for large scale manufacturing of membrane like tissue. Thepreferred size be may be similar to textiles used cloth manufacturing. Acontinuous or batch processing methods may be used. The thickness ofmembrane like scaffold may vary from 1 microns to 3000 microns, morepreferably from 1 microns to 2 mm. The scaffold could also be circular,rectangular, triangular, hexagonal or any other 2-dimensional shape.Square or rectangular shape is preferred due to its simplicity in largescale manufacturing. Several membrane like scaffolds may be stacked in abiotechnology tissue engineering reactor and seeded with mammalian cellsto generate large quantity of engineered tissues suitable for largescale bioprosthesis manufacturing. The scaffold could also have simpleor complex 3 dimensional shapes or geometries. These geometries mayinclude but not limited are: hollow cylindrical tube, cylinder, sphereor complex shapes. Membrane shaped tissue engineered tissue may also becreated by using a hollow cylindrical tube like scaffold and thencutting the tube shaped engineered tissue to make a sheet or membranelike tissue.

Chemical compositions of tissue engineered tissue may be changed bychoosing appropriate cell line or combination of cells in building adesired engineered tissue. The cells could be obtained from manyanimals. Cells from mammalian source are most preferred. The cells maybe obtained form animals source which include animals but not limitedto: human, cow, pig, sheep, deer, ostrich, crocodile, rabbit, rat, snakeand the like. Human, pig, sheep or bovine cells are most preferred. Manytypes of cells could be used to produce a tissue engineered material.The preferred cells chosen which produce large amount of extracellularmatrix proteins such as collagen, elastin and keratin and the like. Thecell types that can be used but not limited to include: fibroblastscells, endothelial cells, smooth muscle cells, muscle cells, cells foundin heart tissue or pericardial membrane, cells found in human or animalskins, sub-mucosa cells, epidermal cells, epithelial cells, and thelike. The cell may be may be purchased from commercial sources such asATCC along with their cell culture media or may be obtained from animalsusing surgical biopsy technique, separated and cultured using knowntissue culture techniques. For example, if a collagen rich tissue isdesired, mammalian cells that produce pericardial type tissue may beused. For skin tissue, cells that generate a combination of collagen andelastin may be used. Stem cells which can be converted into any organmay also be used. Two or more cell types may also be used to create adesired tissue. For example animal cells such as bovine or porcine cellsmay be used to create a structural part of the tissue and the outerlayer may be derived from human cells to give feel, texture andbiological properties of a human tissue. Genetically modified cells mayalso be used to influence the properties of the engineered tissue.

Cell culture medium that is used to grow cells may include severalchemical substances that support the growth of mammalian cells. Cellculture medium may be obtained from commercial sources such asSigma-Aldrich. These substances include chemicals that have beendeveloped or yet to be developed but not limited to are: water, pHcontrolling buffers such as phosphate buffer, HEPES buffer; amino acids;sugars; antibiotics; pH indicator dyes; proteins such as albumin; growthfactors such as fibroblast growth factors; blood products such as serumand the like Cell lines used to grow the tissue will dictate the choiceof cell culture medium and incubation period. Cell culture medium thatdoes not have serum components (serum free medium) is most preferred forlarge scale manufacturing operations. The cell culture mediumcomposition may be used to influence the texture and other properties ofthe engineered tissue. For example, non-natural aminoacid may be used toinfluence the chemical composition of the extracellular matrix producedor cells may be genetically modified to produce a given tissue type. Theengineered tissue may be decellularized prior to medical device use.This step is particularly useful if non-human derived cells are used tomake the engineered tissue.

The tissue generated by tissue engineering methods described above maybe decellularized to generate the extracellular matrix suitable forimplantation. Many methods of decellualrization are known in the art andcould be used without limitation. The preferred methods of tissuedecellualrization are described in the experimental section. Tissue mayalso be crosslinked or chemically modified to improve tissue properties.Some preferred crosslinking methods are described in this invention.Other conventional crosslinking methods such as glutaraldehydecrosslinking, EDC crosslinking, dye mediated photooxidation,hexamethylene diisocyanate crosslinking, crosslinking and the like mayalso be used. The tissue generated by tissue engineering methods may berendered biostable or biodegradable my many methods known in the art ormethods described in this invention. For example, tissue treated withglutaraldehyde, hexamethylene diisocyanate, photooxidation may renderthe tissue substantially biostable. In applications where biodegradabletissue is needed, uncrosslinked tissue or crosslinking catalyzed by-ethyl-3-(3-dimethylaminopropyl carbodiimide) hydrochloride (EDC) ismost preferred The EDC crosslinking is generally known as “zero lengthcross-linking” in the protein modification chemistry art. EDC is a classof compounds generally known as carbodiimides. Carbodiimides generallypromote reaction between carboxylic acid-amine and carboxylicacid-hydroxyl groups to form amide or ester bond respectively. Watersoluble carbodiimides are most preferred crosslinkers. Water solublecarbodiimide such as EDC is most preferred. Other carbodiimides that canbe used include but not limited to:1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide;1-(3-dimethylaminopropyl)-3-ethylcarbodiimide;1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate;1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and the like. EDC cancatalyze a reaction in water over a wide pH range. The pH range may varyfrom 1 to 9. Most preferred pH range is 5 to 7. The desired pH may beachieved by using a biocompatible buffering agents. The preferredbuffers that can be used include, but not limited to, phosphate bufferedsaline (PBS) (pH 7 to 7.5), morpholino ethanesulfonic acid (MES) (pH 5.5to 6.5) and triethanol amine buffer (pH 7 to 7.5), sodium acetate bufferand the like. A buffer concentration in the range of 10 mM to 100 mM ispreferred. Among the buffers, PBS or MES buffers are most preferred. Aco-catalyst that may accelerate the crosslinking reaction is added.Examples of such co-catalyst include, but not limited to,n-hydroxysuccinimide or n-hydroxysulfosuccinimide. The molarconcentration of co-catalyst is in the same range as that of EDC. Thecross-linking reaction is generally completed with in 1 to 600 minutes,more preferably between 1 to 30 minutes. The preferred reactiontemperature is below the shrink temperature of tissue, typically 0 to45° C. Crosslinking reaction at 4 to 37° C. temperature range is evenmore preferred.

The crosslinked tissue is then used to manufacture bioprostheses such asheart valve, stent grafts, vascular grafts, hernia surgical repair,rotating cuff repair and the like. If desired, smaller engineered tissuesizes may be stitched together to make a larger size tissue or thinnertissue may be combined or stacked and glued to make a thicker tissue fora particular medical device application. The engineered tissue may becrosslinked using several methods known in the bioprosthesis processingart or methods yet to be developed. If desired, smaller engineeredtissue sizes may be stitched together to make a larger size tissue orthinner tissue may be combined or stacked and glued to make a thickertissue for a particular medical device application. The engineeredtissue may be crosslinked using several methods known in thebioprosthesis processing art or methods yet to be developed

Medical Applications of Tissue Materials

Tissue prepared according to an embodiment of the present invention may,for example, and without limitation, be used for variety of medical andsurgical applications, e.g., implantable tissue or for manufacture ofbioprosthesis as defined earlier. These applications include, but arenot limited to, applications which are already known such as, by way ofexample, and not limitation, vascular patch, heart valve, venous valvebioprosthesis, vascular grafts, surgical patch, or applications whichare yet to be developed. Complex medical devices such as, by way ofexample, and not limitation, heart valve, stent grafts and sewing cuffsare also included. Many variations of valve designs may, for example,and without limitation, be used. The tissue used may be subjected toadditional anticalcification treatments such as, by way of example, andnot limitation, treatment with ethanol solutions (>50% buffered ethanolsolution) or surfactants or chemical modifications with long alkylchains. Medical devices such as, by way of example, and not limitation,coronary stents or peripheral stents may be partially or fully coveredwith the tissue such as, by way of example, and not limitation, bovinepericardial or porcine pericardial tissue or porcine sub-mucosa tissueand used. In one embodiment, the present invention provides for a methodfor making a tissue/stent graft including the steps of providing anexpandable metal or polymeric stent capable of supporting mammalian cellgrowth on its surface; exposing the stent to a mammalian cell culturemedium including live mammalian cells capable of attaching and growingon the stent; growing the cells on the stent surface until the stentsurface is partially or completely covered by the cells and extracellular matrix; and exposing the stent to a tissue fixative solution.Coated tissue may also be used make stent grafts used in the vascularsurgery. The present invention also encompasses an expandable stentwherein the stent is fully or partially wrapped or covered with anon-cytotoxic membrane-like tissue. Bovine ureter, bovine thoracicartery or other arterial tissue may, for example, and withoutlimitation, be used as small or medium bore vascular grafts. The tissueused may be loaded with biological active compounds by methods known inthe art or methods described herein. Hybrid devices such as, by way ofexample, and not limitation, graft tissue coated with polymericelastomers may, for example, and without limitation, be used to makeimproved vascular grafts. In one embodiment, a polymeric vascular graftmade from polyurethane elastomer may be completely wrapped in a membranelike tissue. Such wrapping provides animal tissue as blood contactingsurface for the graft. The polyurethane provides a self sealing propertyto the graft. In another embodiment, crosslinked bovine thoracic arteryis dehydrated using a series of alcohol solutions and finally usingdimethyl acetamide (DMAC) as a solvent. A 10% solution of Biomer®polyurethane in DMAC is sprayed on the outer surface of the graft. Thesolvent is removed by air drying and finally by vacuum drying. Thepolyurethane-tissue composite graft may offer improved mechanicalproperties.

The animal tissue, especially a membrane-like tissue such as, by way ofexample, and not limitation, porcine pericardial tissue, bovinepericardial tissue or porcine intestinal tissue may, for example, andwithout limitation, be used in wound management applications. The woundmanagement applications include, but are not limited to, venous ulcers,drainage wounds, diabetic ulcers, chronic vascular ulcers, pressureulcers, trauma wounds, surgical wounds, burn wounds, partial and fullthickness wounds and the like. Membrane-type tissue coated withbiodegradable polymer capable of releasing bioactive substances ispreferred. The preferred bioactive substances for wound managementinclude, but are not limited to, growth factors derived from blood suchas, by way of example, and not limitation, PGDF, anti-infectivecompounds like rifampin, and chlorhexidene acetate.

The tissue-based patch may also be used in various surgicalapplications. Depending on the application, a biostable or biodegradabletissue may, for example, and without limitation, be used. In oneembodiment, the present invention provides for a method sealing aleaking tissue during a surgical procedure including the steps ofproviding a membrane like animal tissue suitable for human implantation;providing a surgical adhesive capable of adhering with the tissue; andcuring a surgical adhesive between a leaking site and membrane liketissue. The applications include, but are not limited to providingpubourethral support for the treatment of urinary incontinence;abdominal wall repair; vascular surgery applications like intracardiacdefect repair, septal defect repair, great vessel repair and anulusrepair; suture-line buttressing; hernia repair; perforated tissuerepair; general tissue repair such as, by way of example, and notlimitation, prolapsed tissue repair, thoracic wall repair, carotidpatch, pelvic floor repair, bladder repair; dural substitute for closureof dura during neurosurgery;

Membrane-like tissue (biostable or biodegradable) such as, by way ofexample, and not limitation, bovine pericardium, porcine pericardium,porcine submucosa may be cut into strips, strands or fibers. Many knownmethods to cut the tissue into small fiber-like strands may, forexample, and without limitation, be used. The methods reported in theprior art may, for example, and without limitation, be used. Thesemethods include, but are not limited to, methods such as, by way ofexample, and not limitation, mechanical shredders, ultraviolet, visibleand infrared laser cutting-based methods, water jet cutting methods,ultrasonic wave-based cutting methods and the like. The shredded tissueor fiber may be braided or woven to generate cloth like structures orrope like structures. These braided or woven structures may, forexample, and without limitation, be used to fabricate many devices suchas, by way of example, and not limitation, vascular patch, vasculargrafts, stent grafts etc. Synthetic fibers such as, by way of example,and not limitation, polyester fiber, Teflon® fiber, polyethylene fiber,poly propylene fiber, silk fiber, poly(hydroxy acid) or polylactide orcarbon fiber may be braided or woven along with braided tissue to formcomposite structures. Such composite may provide additional propertiesto the woven or braided structures. Such braided structures may, forexample, and without limitation, be used as substitutes for ligaments.The synthetic fibers used may be biodegradable.

Tubular tissue such as, by way of example, and not limitation, arterialtissues may, for example, and without limitation, be used to makecatheters. For example, catheters that require a longer implantationtime may be made using crosslinked animal tissue. The outer layer ofnerve tissue may, for example, and without limitation, be used to makenerve guide repair devices.

Transparent tissues such as, by way of example, and not limitation,porcine cornea may, for example, and without limitation, be used toobtain a transparent material If unsaturated monomers are used in suchtissue fixation, then monomers which give transparent hydrogel may, forexample, and without limitation, be used. For example, tissue may befixed with 2-hydroxyethyl methacrylate, vinyl pyrrolidinone monomerswhich give optically transparent hydrogels. In general, monomers whichare used in commercial contact lens manufacturing are preferred. Suchtransparent materials may, for example, and without limitation, be usedin various ophthalmic applications such as, by way of example, and notlimitation, corneal transplants, implantable contact lens or intraocularlens, etc. Tissue fixed with a degradable crosslinkers may, for example,and without limitation, be used as a scaffold for tissue engineering.

In many medical applications, implantable tissue may be subjected toadditional crosslinking or anticalcification treatments which are knownin the art. Such alternations, combinations or modifications areencompassed by the present invention even if specific examples orembodiments are not provided in this manuscript.

The membrane-like tissue is especially useful as a barrier forprevention of postoperative adhesions. Membrane tissue coated withnon-cell adhesive hydrogels or polymars such as, by way of example, andnot limitation, polyethylene glycol based hydrogel or PTFE basedmembranes is especially useful. A localized release of cell cycleinhibitor such as, by way of example, and not limitation, Rapamycin,paclitaxel, Actinomycin, Lovastatin through coated or composite tissuemay also assist in reducing postoperative adhesions.

Hernia repair is among the most common general surgery procedures.Complications cited in the medical literature include intestinalfistulas and surgical adhesions, which may cause intestinal obstruction.The coated tissue compositions or tissue-PTFE composite membrane likematerials described in this invention are especially useful for herniarepair applications. A membrane like tissue (biodegradable or biostable)described in this invention or commercially available tissue patch suchas AlloDerm® marketed by LifeCell Corporation is coated withpolyethylene glycol coating on one side is especially useful for Herniarepair application. The PEG coating acts as a non-cell adhesive layerwhich reduces postoperative adhesions. The other non-coated sidepromotes cell adhesion and integrates into the native tissue. The PEGbased coating operation may be carried out prior to implantation. Forexample, AlloDerm® patch may be coated with DuraSeal™ sealant fromConfluent Surgical Inc., Waltham, Mass. just prior to implantation andmay be used for hernia repair applications. The PEG coating may bereplaced by non-cell adhesive membrane like PTFE membrane such asGore-Tex surgical patch which is also known to reduce post-operativeadhesions. The ePTFE membrane-tissue or AlloDerm® composite may beprepared by suturing the two membranes using ePTFE based sutures.

Membrane-like tissue such as, by way of example, and not limitation,bovine pericardium or porcine pericardium may, for example, and withoutlimitation, be used as structural backing material for tissue adhesives.Many types of surgical adhesives could be used including those that arecommercially available or yet to be developed. In one approach,structural adhesive material or their precursors may be coated on oneside of pericardial tissue. The tissue provides the mechanical supportfor the adhesive material. Upon application, adhesive materials developstrong adhesive bond between surgical site and tissue backing materialused. The surgical adhesive and pericardial tissue could bebiodegradable or biostable. In one exemplary approach, a 2 cm by 5 cmnon-crosslinked pericardial tissue or sub-mucosa tissue is coated with aliquid surgical adhesive such as, by way of example, and not limitation,a photopolymerizable liquid diacrylate along with a visible lightphotoinitiator (see Example 35 described below) and cured between a lungtissue and pericardial tissue to seal air leaks. In another approach,cynaocaylic acid ester is used as a adhesive. Many cynoacrylateadhesives could be used these include ester already known in the art orester yet to be develop for medical device applications. The preferredcynoacrylate esters include but not limited to: methyl, ethyl, butyl oroctyl ester and is cured between a lung tissue and uncrosslinkedpericardial tissue. Butyl cynoacrylate may also be substituted withFocalSeal® surgical adhesive available from Genzyme Biosurgery. Becauseuncrosslinked pericardial tissue and polybutyl cynoacrylate aredegradable, the patch and adhesive combination serves as a degradableadhesive patch system. In another exemplary approach, polyethyleneglycol n-hydroxysuccinimide based crosslinker (PEGNHS) and amine basedcrosslinking agent are mixed and coated on the tissue in dry state. Uponhydration, the PEHNHS and amine crosslinker react to form a crosslinkedpolymer and form a strong adhesive bond. Other surgical adhesives orglues such as, by way of example, and not limitation, albumin glue mayalso be used to make an adhesive patch.

Porcine/bovine meniscus tissue modified according to an embodiment ofthe present invention may also be used in meniscus repair procedures.

Catheters or catheters parts may be made from implantable animal tissue.Catheters designed to stay with in the body for 3 days or more may bemade using compositions according to an embodiment of the presentinvention.

Membrane-like tissue (biodegradable or biostable) may, for example, andwithout limitation, be used to fabricate medical devices such as, by wayof example, and not limitation, tissue coated stents or stent-grafts.The tissue covering on the stent may be done from outside, inside orform both sides. The stent may be a vascular stent such as, by way ofexample, and not limitation, coronary stent or peripheral stent ornon-vascular stent. Stent used may be biodegradable (made formbiodegradable polymers such as, by way of example, and not limitation,cyclic polylactones and their copolymers) or biostable such as, by wayof example, and not limitation, metallic stent (made from stainlesssteel or Nitinol). The tissue used on the stent may be loaded with drugssuch as, by way of example, and not limitation, cell cycle inhibitorswhich are known to inhibit restenosis.

In minimally invasive surgical applications thin but strong membranetissue is desired. Membrane-like tissue such as, by way of example, andnot limitation, bovine pericardium may be too thick in MIS surgicalapplications. In stent graft application, a strong membrane-like tissuewhich is about 10 to about 1000 micron thick is highly desirable. Suchthin tissue reduces the profile for a stent graft delivery systemtherefore permits its use in making small diameter (low profile)devices. The thickness of the tissue can be reduced by many methodsknown in the art such as, by way of example, and not limitation,cutting, polishing and the like. The most preferred method forcontrolling the thickness is laser-based ablation technique used invision correction surgery such as, by way of example, and notlimitation, photorefractive keratectomy or IntraLase® surgicaltechnique. Laser-based reshaping of tissue is routinely used in Lasiksurgery, short for laser-assisted-in-situ keratomileusis, normally usedin vision correction surgery. The membrane-type tissue such as, by wayof example, and not limitation, porcine submucosa or porcine pericardiumor bovine pericardium tissue can be treated using Lasik-like surgery toreduce the desired tissue thickness. Lasik-like technique may, forexample, and without limitation, be used to reduce the tissue thicknessto about 5 to about 500 microns, most preferably about 50 to about 500micron thick tissue without significantly compromising other propertiesof the tissue.

The coated tissue described in this invention may be useful to formstrip-like structures that can be used in bone repair. For example,biodegradable tissue like porcine or bovine pericardium may be loadedwith bone growth factors such as, by way of example, and not limitation,BMP-2 or BMP-7. The BMP loaded tissue may, for example, and withoutlimitation, be used to heal bone surface defects. Multiple layers ofsuch structures may, for example, and without limitation, be used inbone fracture repair. The membrane-like porcine pericardium tissue maybe coated with dematerialized bone matrix. The membrane tissue providesstructural strength while demineralized bone matrix provides growthfactors and matrix that promotes bone growth. Such composite materialsmay, for example, and without limitation, be used as a “bonewrap” inbone fracture repair. In another approach, the pericardial tissue may becoated with demineralized bone matrix particles and the composite may,for example, and without limitation, be used as a bone wrap.

Tissue hydroxyapatite composite matrix may be used in bone repairapplications. The hydroxyapatite or other bone promoting calcium saltsis added to the tissue matrix using many methods known in theorthopaedic biomaterials art. In one exemplary embodiment, hydroxyapatite is synthesized inside the tissue matrix by combining calcium ionand phosphate ion solution in presence of tissue. The calcium nitrate[Ca(NO3)2, 4H2O, Aldrich], may be used as calcium source and ammoniumhydrogen phosphate [(NH₃)₂HPO₄, Aldrich] may be used as phosphorussource. In presence of tissue, the calcium nitrate solution is added toammonium hydrogen phosphate solution at 40° C. while maintaining the pHusing ammonium hydroxide to 10 and 10.6 to form hydroxyapatite crystals.The reaction mixture is stirred for 24 hours to stabilize the crystals.The ratio of The Ca ion and P ion precursors is maintained 1.6 to makehydroxyapatite crystals. This reaction is carried in presence of tissuesuch as pericardial tissue so that hydroxyapatite is generated in sidethe tissue. It is contemplated that the tissue-hydroxyapatite canpotentially be used as a matrix for bone formation. If needed bonepromoting bioactive compounds such as bone morphogenic proteins (BMP)may be added in the composite matrix to accelerate the bone formation.

In a biodegradable drug delivery patch application, membranes liketissue such as, by way of example, and not limitation, porcinepericardial tissue, bovine pericardial tissue or porcine sub-mucosa,porcine bladder tissue are preferred. The animal tissue used in thepatch application may be crosslinked or non-crosslinked. Anon-crosslinked tissue is used if an absorbable patch material desired.Drug delivery patch could be formulated to incorporate bioactivecompounds. The patch will deliver a therapeutically effective amount ofbioactive substance in a controlled manner. The patch may be coated withbiodegradable hydrogels encapsulated with cells or bioactive growthfactors.

In one potential application, an animal tissue-based patch is used toinduce angiogenesis in the heart. Briefly, an animal tissue-based patchis formulated to deliver vascular endothelial growth factor (VEGF) in acontrolled manner. The patch is then transported to a surgical site inthe body such as, by way of example, and not limitation, the surface ofa beating heart and immobilized on the heart surface. The localizedrelease of VGEF induces angiogenesis in the heart. In anotherapplication, a patch is formulated to release cell cycle inhibitor suchas, by way of example, and not limitation, Rapamycin, Paclitaxel,Actinomycin, Lovastatin, and the like. The patch is then transported tothe site where vascular stent or graft is implanted to a site that issusceptible for restenosis. The patch is wrapped around the vessel whererestenosis is likely to occur and cell cycle inhibitor is releasedlocally. The local release of cell cycle inhibitor reduces or eliminatesrestenosis. In some applications, a patch may be coated with hydrogelcoatings containing living species such as, by way of example, and notlimitation, mammalian cells, stem cells, and endothelial cells. Afterimplantation, the cells may perform a therapeutic function. Cellencapsulation methods such as, by way of example, and not limitation,those described in U.S. Pat. No. 5,529,914, which cited here forreference only as one suitable technique, may be preferentially used incell encapsulation.

Some of the tissue modifications or crosslinking methods may beperformed on autologous tissue obtained during a surgical procedure.Chemicals and instrumentation, and tools required for such proceduresmay be provided as a kit in the operating room. Autologous or singletissue may, for example, and without limitation, be used as a carrierfor biologically active compounds. The drug-tissue based combination maybe prepared in the operating room during a surgical procedure. A kitincluding biodegradable polymer such as, by way of example, and notlimitation, polylactic acid/polyglycolic acid copolymer, a non-toxicbiocompatible preferably water soluble solvent capable of dissolvingbiodegradable polymer is provided according to an embodiment of thepresent invention. The preferred solvents that can be used include, butare not limited to, n-methylpyrrolidinone, dimethyl sulfoxide, acetone,polyethylene glycol, and the like. The biodegradable polymers include,but are not limited to, polymers, copolymers or oligomers of glycolide,dl-lacide, d-lactide, l-lactide, caprolactone, dioxanone andtrimethylene carbonate; polyhydroxyacids, polylactic acid, polyglycolicacid, polyanhydrides, polylactones, polyhydroxyalkanoates which arepolyesters produced by microorganisms such as poly(3-hydroxybutyrate),3-hydroxyvalerate, 4-hydroxybutarate, 3-hydroxyhexanoate,3-hydroxyoctanoate. Preferred hydrophilic polymers include, but are notlimited to, polyethylene glycol-polyhydroxy acid or polyethyleneglycol-polylactone copolymers (PEG-PL copolymers), polyvinyl alcoholco-polylactone copolymers, and derivatives of cellulose, hyaluronic acidand dextran. The PEG-PL copolymers are most preferred. PEG-PL copolymerssuch polyethylene glycol-polylactate copolymers can have a range ofproperties from hydrophobic to hydrophilic depending on the amount ofPEG incorporation in the copolymer and molecular weight of PEG andpolylactone. The biodegradable polymer solution may be made in theoperating room from the sterile components. The biologically activecompound is dissolved, suspended or dispersed in the polymer solution.An autologous tissue piece is cut using a surgical incision and thenincubated in the polymer solution. The tissue is then removed from thesolution and washed with saline and implanted. The polymeric coatingholds and releases the bioactive compounds in a controlled manner. Forexample, rifampin, an antibiotic may be released locally to controlinfection. The autologous tissue serves as a matrix that can be suturedto immobilize it at a localized disease site. The low toxicity ofsolvents and biodegradable nature of polymer used in this system providea simple method for preparing a tissue-based controlled drug deliverydevice.

The present invention also provides for a method for localized celltherapy including the steps of providing or obtaining a sterile tissuesuitable for human implantation; providing a sterile crosslinkablepolymer; providing a sterile aqueous based solvent for the crosslinkablepolymer; providing or obtaining mammalian cells for therapeutic use;dissolving the polymer in the solvent and dispersing cells in thepolymer solution; forming a coating of polymer on the tissue andcrosslinking the polymer without damaging the cells; and implanting thetissue.

It is contemplated that the shape memory capable tissue may be used tomake many novel medical devices. For example, tissue based vasculargrafts may be compressed and the compressed shape may be preserved toimprove kink resistance of the graft. The compressed graft becomelongitudinally compliant as a result of compression and may have betterpatency rates. A heart valve leaflet shape may be preserved to reducestresses during heart valve device use. A tissue based breast implantwith appropriate shape may be preserved using methods described in thisinvention.

The selective modification of tissue to create biodegradable andbiostable regions may be used to create new medical devices.

In many medical device applications, bioprostheses prepared may besterilized by many methods known in the art. These methods include butnot limited are: gamma radiation sterilization, ethylene oxide orpropylene oxide based sterilization methods, electron beamsterilization, iodine-based sterilization methods, and the like. Thepreferred method is ethylene oxide gas sterilization or gamma radiationsterilization. The sterilization process parameters will depend on thetissue based medical device to be sterilized.

In some applications proteins such as, by way of example, and notlimitation, collagen or albumin are treated with silver salt solution toform silver salts/complexes of amino acids present in the proteins. Forexample, aspartic acid which produces free carboxyl acid groups in theproteins may be treated with silver to form a silver salt. The resultantsilver salt then releases silver ions upon implantation producing anantimicrobial effect due to silver ions.

The tissue-based medical devices described herein may be packaged usingvariety of packaging methods including packaging in a liquid solutionsuch as, by way of example, and not limitation, glutaraldehyde solution.In preferred packing method, the tissue is packaged in sealed plasticbag. If desired, the tissue may be packaged and stored under inert gasatmosphere such as, by way of example, and not limitation, nitrogen,carbon dioxide or argon gas. The gas used may be kept under pressure.

The following non-limiting examples are intended to illustrate theinventive concepts disclosed in this document. Those skilled in the artwill appreciate that, in light of the teachings of the presentinvention, modifications can be made to these examples, drawings,illustrations and claims which are contemplated to fall within the scopethe present invention.

Materials and Methods

Tissue-like bovine pericardium, porcine pericardium, porcine sub-mucosa,porcine aortic root, porcine meniscus tissue and bovine thoracicarterial tissue are acquired from local abbotair or slaughter house. Theanimals from abbotair or the tissue obtained form abbotair may bescreened or tested for infectious viruses, bacteria or proteins such as,by way of example, and not limitation, BSE which cause disease such as,by way of example, and not limitation, Bovine Spongiform Encephalopathy.Tissue infected with harmful virus or proteins may be discarded.Preferably, the tissues are collected from slaughter house with in 30minutes after the kill. Within 36 hours, the tissue collected is cleanedto remove excess fat, connective tissue, blood and other cellularcomponents and rinsed with cold phosphate buffered saline and stored inhyperosmotic storage solution (ESWHS solution) until fixation or othermodifications. In many instances, the tissue may be treated withdetergents, organic solvents such as, by way of example, and notlimitation, aqueous ethanol or long chain alcohols such as, by way ofexample, and not limitation, octanol or 1,2-octane diol other tissuecleaning solutions that remove cells/cell debris from the tissue andleaves behind only extracellular matrix. The acellular extracellularmatrix is preferred in many medical device applications. Acrylic acidn-hydroxysuccinimide ester, acetic acid n-hydroxysuccinimide,polyethylene glycol n-hydroxysuccinimide esters, acrolein are purchasedfrom Sigma-Aldrich or Pierce or Sherewater. Polyethylene glycol can bepurchased form various sources such as, by way of example, and notlimitation, Nektar Therapeutics (formerly Shearwater Polymers), DowChemicals (Union Carbide), Fluka and Polysciences. Multifunctionalhydroxyl and amine terminated polyethylene glycol are purchased fromNektar Therapeutics, Dow Chemicals and Texaco Amine-terminatedpolyethylene glycols also can be synthesized using methods known in theprior art. Other specialized polyethylene glycol derivatives may also bepurchased or custom synthesized from Nektar Therapeutics. DL-lactide,glycolide, caprolactone and trimethylene carbonate can be obtained fromcommercial sources like Purac, DuPont, Polysciences, Aldrich, Fluka,Medisorb, Wako and Boehringer Ingelheim. N-hydroxysulfosuccinimide canbe purchased from Pierce. All other reagents, solvents are of reagentgrade and can be purchased from commercial sources such as, by way ofexample, and not limitation, Polysciences, Fluka, ICN, Aldrich andSigma. Most of the reagents/solvents are purified/dried using standardlaboratory procedures such as, by way of example, and not limitation,described by Penin et al. Monomers may be purified to remove inhibitorsjust prior to use. For example, liquid monomers such as, by way ofexample, and not limitation, 2-hydroxyethyl methacrylate may bevacuum-distilled to remove the inhibitor. Most commercially availablemonomers are purchased from Polysciences, Fluka, ICN, Aldrich and Sigma.Photoinitiator2-Hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone (Irgacure2959) and 2,2-dimethoxy-2-phenylacetophenon (Irgacure 651) are purchasedfrom Ciba Specialty Chemicals or Sigma-Aldrich. Small laboratoryequipment and medical supplies can be purchased from Fisher orCole-Parmer. Cell culture experiments are performed using a standardmammalian tissue culture laboratory or microbiology laboratory capableof handling and growing mammalian and human cell cultures.

Removal of Cellular Components from the Tissue (TissueDecellualrization)

Tissue suitable for implantation may be treated to remove cells andcellular components or fragments, DNA fragments and the like. Theremoval of cellular components is believed to reduce immunogenicity ofthe implanted tissue. Upon removal of cellular components, onlyextracellular matrix components such as, by way of example, and notlimitation, collagen, elastin, laminin, keratin, glycosaminoglycans areretained in the tissue. The extracellular matrix is believed to benon-immunogenic. Many protocols for removal of cellular components arereported in the prior art, one of the preferred protocols is providedbelow. Ten 1 cm by 1 cm bovine pericardium pieces, cut from a freshlyobtained bovine pericardial sac, are transferred to 50 ml conical flaskcontaining 10 ml phosphate buffered saline (PBS, pH 7.2). The tissue isthen incubated in 100 ml distilled water at 4° C. for 48 h, followed byincubation in 30% sodium chloride and/or saturated sodium chloridesolution for 48 hours followed by washing with 100 ml PBS solution threetimes. The tissue is then treated with 40 ml of 0.25% trypsin-EDTA, for30 minutes at 37° C.; followed by washing with warm (37° C.) 100 ml PBSsolution three times. It is then treated with 40 ml of micrococcalnuclease, 1 unit/ml, for 20 minutes at 37° C.; followed by washing withwarm (37° C.) 100 ml PBS solution three times. It is then furthertreated with 40 ml of Triton X-100, 10%, for 10 minutes at 37° C.;followed by washing with warm (37° C.) 100 ml PBS solution three times.It is then treated with 40 ml of 0.1 M sodium hydroxide at 37° C. for 30minutes; followed by washing with warm (37° C.) 100 ml distilled waterfive times. It is then treated with 40 ml of 0.1M hydrochloric acid at37° C. for 30 minutes; followed by washing with warm (37° C.) 100 mldistilled water five times. Finally the tissue is washed with 70%isopropanol three times and stored in 70% isopropanol at −20° C. untilfurther use.

In a simplified cell removal protocol (tissue decellualrizationprotocol)), tissue is first treated with a 0.25% trypsin-EDTA solutionin PBS for 30 minutes at 37° C. followed by a treatment with 10%surfactant solution (Triton X-100) for 2 hours to remove cellular debrisor soluble proteins.

Shrink Temperature by Differential Scanning Calorimetry

Shrink temperature is evaluated by differential scanning calorimetry.Briefly, 3-10 mg of tissue sample is heated in a sealed aluminum samplepan and heated at 10° C. per minute up to 200° C. under nitrogenatmosphere. The endotherm around 55° C. to 110° C. is attributed asshrink temperature.

Shrink Temperature by Visual Method

A 3 cm×1 cm tissue strip is cut and the rectangular shape of the tissueis traced on a paper to record its size. In a 500 ml beaker, 300 mlsaline solution is transferred. The tissue is then suspended in thesaline solution using a cotton or metal thread and is completelyimmersed in the saline solution. The solution is slowly heated on a hotplate until boiling. The tissue is visually observed until signs forshrinkage. The temperature at which tissue significantly shrinks isrecorded. The tissue is removed from the solution, washed with water.The size of the tissue again traced on a paper and compared with theoriginal size. The shrunken tissue shows significant reduction in size(typically >20% shrinkage). Untreated tissue and treated tissue areusually compared in the same experiment.

Viability and Spreading of Bovine Endothelial Cells on CrosslinkedBovine Pericardial Tissue

Tissue samples fixed using crosslinking agents such as, by way ofexample, and not limitation, photopolymerization, are used in thisexperiment. Untreated bovine pericardium tissue is used as control. 1cm×1 cm size tissue samples are soaked in 70% ethanol (overnight),washed with sterile PBS (15 minutes, 3 times) and soaked in MinimumEssential Medium (MEM) supplemented with aminoacids, antibiotics and 30%fetal bovine serum (FBS) for 1.5 h. The tissue samples are thentransferred to a 24 well sterile tissue culture plate. Bovine aorticendothelial cells passaged in MEM/10% FBS are resuspended in MEM/10% FBSand approximately 200000 cells are carefully placed on the tissuesamples. The cells are allowed to adhere for 30 minutes before adding0.75 ml of the same medium to each sample. After 24 hours of incubationin a 5% carbon dioxide atmosphere at 98% humidity and 37° C., thesamples are transferred to new wells and fresh medium is added to thesewells. The samples are incubated for a further period of 24 to 72 hours.At the end of this incubation period, the tissue samples/cells arewashed with PBS (3 times, 5 minutes per dish at room temperature) andfixed using 4% formaldehyde for 10 minutes. After rinsing the samples inPBS as described above, they are treated with 0.1% Triton X100 in PBSfor 3 minutes. The cells are then stained with phalloidin/rhodamine(diluted 1:40 in PBS) in dark for 45 minutes, rinsed 3 times in PBS andviewed immediately under a fluorescence microscope. The morphology andcoverage of endothelial cells on tissue samples are recorded. Somerepresentative snapshots are photographed for each sample.Glutaraldehyde-treated tissue is used a negative control and untreatedtissue is a positive control. The untreated and photopolymerizationtreated tissue (vinyl pyrrolidinone treated) showed substantially highercell growth and attachment as compared to glutaraldehyde fixed tissue.It is generally known that glutaraldehyde fixed tissue does not supporttissue growth.

Subcutaneous Implantation of Tissues

1 cm2 rectangular tissue pieces are implanted using a standard surgicalprotocol. Prior to implantation, the tissues are rinsed for 3 minutes ineach of three basins containing 500 ml of sterile PBS, accompanied bygentle shaking. Specimens are implanted subcutaneously 1 cm lateral fromthe abdominal midline in 3 week old Sprague-Dawley rats and retrievedafter 60 days. Unimplanted samples (two per crosslinking treatment) areused as unimplanted control. Untreated tissue is used as positivecontrol. Glutaraldehyde-fixed tissue is used as a negative control.Tissue modified with unsaturated group and infused with monomer andphotoinitiator but not exposed to light is treated as dark control.

X-Ray Imaging and Histology

The subcutaneously implanted samples are recovered after 30, 60 or 90days. The explanted samples are subjected to standard medical x-rayimaging for calcification/mineralization as a result of implantation.The unimplanted samples and samples fixed with glutaraldehyde are alsoimaged and used as controls. The calcified deposits can be seen insamples treated with glutaraldehyde. An untreated unimplanted sampledoes not show calcific deposits when viewed using medical x-ray imagingtechnique or when stained using Van Kossa stain. After visual analysis,the parts of the samples are sectioned, stained with H&E, Masson'strichromeand and Van Kossa stain and then evaluated histologically forinflammation, vascularization, calcification and collagen organization.Inflammation consists of aggregates of lymphocytes and plasma cells, andthe presence of mast cells in the implants and is graded from 0=None, to5=Severe (most of the section with large lymphoid aggregates).Calcification is also graded from 0 to 5 based on Van Kossa stain.

Calcium Assay

The calcium content of the subcutaneously implanted samples isdetermined by the wet-washing technique. Briefly, up to 0.5 g ofexplanted tissues are dried and weighed and by heated up to 250° C. with4 ml of concentrated sulfuric acid followed by heating up to 300° C.with addition of concentrated nitric acid. The resulting digested sampleis brought to 40 ml with addition of high purity water. Calciumconcentration in the solution is determined by standard ICP methodology.

Pepsin Digestion Assay-Analysis of Tissue Fragments by GelElectrophoresis

Pepsin digestion is done to evaluate the ability of tissue to resistenzymatic degradation. Briefly, the fixed and control tissue samples areexposed to a pepsin solution and size and weight of the tissue ismonitored. Untreated and uncrosslinked tissue sample is used as acontrol. In another method, tissue samples are clipped into smallfragments and digested for 4 h at 37° C. in 10 mM HCl solutioncontaining 4 mg/ml pepsin. Sham digestions are also prepared in HClwithout the enzyme. Following the incubation, the samples arecentrifuged at 4° C. for 1 hour at 14,000 rpm. The supernatants areanalyzed by gel electrophoresis using 10-20% acrylamide:bisacrylamide(37.5:1) gradient gel. Control samples with the same quantity of enzymeare also treated as above, but in the absence of tissue. The fixedtissue shows significantly lower proteins on gel as compared to unfixedtissue. The fixed tissue also shows lower weight loss when compared tounfixed tissue indicating stability of the tissue.

Pepsin Digestion Assay-Gravimetric Analysis

The tissue to be treated is washed with distilled water and incubated indistilled water for 10 minutes. The same is then dried in air and thenkept in vacuum oven for drying until constant weight is observed. Thedried tissue is then incubated in 10 mM HCl containing 40 mg of pepsinfor 12 hours. The tissue is removed, washed with distilled water andagain dried till constant weight. The difference in the tissue weightsbefore and after the test is noted. The loss in weight shows the amountof protein digested by pepsin. The unfixed or uncrosslinked tissuegenerally shows substantial loss as compared to fixed or crosslinkedtissue.

Collagenease Digestion Assay

The resistance to enzymatic digestion also can be found by exposing thetissue to collagenease solution. Briefly, a tissue is exposed to a 0.01mg/ml collagenease enzyme solution in 10 mM TES buffer(N-tris[hydroxymethyl]methyl-2 aminoethane sulfonic acid) and 25 mMcalcium chloride solution at 37° C. for 24 to 96 hours and the weightloss of the tissue is monitored and compared with the control untreatedtissue.

Free Amine Content of Tissue

The free amine content of the treated tissue is determined by theninhydrin assay. Briefly, tissue is exposed to ninhydrin solution andthe absorbance of ninhydrin solution is measured at 570 nm and iscompared with control tissue (untreated uncrosslinked tissue). Theabsorbance is normalized by dividing by weight of tissue beforecomparison.

Biodegradation and Biocompatibility of Tissue and Tissue-BiodegradablePolymer Composites

In vitro degradation of the polymers is followed gravimetrically at 37°C., in aqueous buffered medium such as, by way of example, and notlimitation, phosphate buffered saline (pH 7.2). In vivo biocompatibilityand degradation life times are assessed after subcutaneous implantationof tissue samples. The implant is surgically implanted in the animalbody. The degradation of the implant over time is monitoredgravimetrically or by chemical analysis. The biocompatibility of theimplant is assessed by standard histological techniques.

General Analysis

Chemical analysis such as, by way of example, and not limitation,structural determination is done using nuclear magnetic resonance(proton and carbon-13) and infrared spectroscopy. High pressure liquidchromatography or UV-visible spectrophotometry is used to determine drugelution profiles. Gel permeation chromatography is used for molecularweight determination. Thermal characterization such as, by way ofexample, and not limitation, melting point and glass transitiontemperature is done by differential scanning calorimetric analysis. Theaqueous solution properties such as, by way of example, and notlimitation, self assembly, micelle formation, and gel formation aredetermined by fluorescence spectroscopy, UV-visible spectroscopy andlaser light scattering instruments.

Example 1 Modification of Tissue Using Unsaturated Succinimide Ester(Acrylic Acid Succinimide Ester)

Modification of Bovine Pericardium Tissue.

Ten 1 cm by 1 cm bovine pericardium pieces, cut from a freshly obtainedbovine pericardial sac, are transferred to 50 ml conical flaskcontaining 10 ml phosphate buffered saline (PBS, pH 7.2). 250 mg acrylicacid succinimide ester (ANHS) (Sigma-Aldrich Product Number: A8060)dissolved in 0.5 ml dimethyl sulfoxide is added to the conical flask andthe solution is vortexed for 15 minutes. 0.1 g of ANHS in 0.1 ml DMSO isadded to the fixation solution every 2 hours up to six hours. Themodification reaction is carried for 6 hours at ambient temperature (25°C.) and then for 12 hours at 4° C. with gentle shaking. The reaction isterminated by washing the tissue with 20 ml PBS 3 times. Finally, thetissue is stored in 30 ml 38% isopropanol and 2% benzyl alcohol solutionat 4° C. until further use.

In another variation of this method, porcine pericardial sac is obtainedfrom a local abbotair and is cleaned to remove blood and fatty tissuecontaminants from the tissue surface. Ten 1 cm by 1 cm pieces are cutfrom the cleaned pericardial sac and transferred to 10 ml 0.20 M2-(N-morpholino)ethanesulfonic acid (MES) buffered solution (pH 6.5).0.5 g acrylic acid n-hydroxy succinimide ester (ANHS) is added to thebuffered solution and the reaction is vortexed for 15 minutes andcontinued for 6 hours at ambient temperature. An additional 0.1 g ANHSester is added to the mixture at every 1.5 hours. After the reaction,the tissue is isolated and washed several times with PBS and finallystored in 40% isopropanol solution.

Modification of Arterial and Porcine Heart Valve Tissue.

One 1.5 cm piece of bovine thoracic artery and one third section ofporcine aortic root containing one valve cusp and one attachment zoneand wall is transferred to 50 ml polypropylene centrifuge tubecontaining 10 ml PBS. 250 mg of acrylic acid succinimide ester dissolvedin 0.5 ml dimethyl sulfoxide is added to the tube and the solution isvortexed for 15 minutes. The modification reaction is carried for 6hours at ambient temperature (25° C.) and then for 12 hours at 4° C.with gentle shaking. The reaction is terminated by washing the tissuewith 20 ml PBS 3 times. Finally, the tissue is stored in 30 ml 40% HEPESbuffered isopropanol solution.

Modification of Ligament and Meniscus Tissue.

Using a similar procedure described above 1 cm ligament tissue and 1 cmby 1 cm porcine meniscus tissue is modified by 250 mg of acrylic acidsuccinimide ester dissolved in 0.5 ml dimethyl sulfoxide.

Modification of Corneal Tissue.

Porcine cornea is obtained from a local abbotair and is washed with PBS(pH 7.2) to remove blood and other biological contaminants. The corneais placed in 100 ml beaker containing 50 ml PBS solution. To thissolution, 0.2 g acrylic acid succinimide ester dissolved in 2.0 mldimethyl sulfoxide is added. The reaction continued for 12 h at ambienttemperature. The tissue is removed, washed several times with PBS andstored until use. The reaction conditions are chosen to maintain thetransparency of corneal tissue (minimum change in refractive index).

Example 2 Modification of Tissue Using Glycidyl Methacrylate

10 pieces of 1 cm by 1 cm bovine pericardium tissue are transferred to100 ml conical flask containing ten ml PBS and a magnetic stir bar. Tothis solution, 2 ml glycidyl methacrylate, 2 ml triethyl amine and 0.5 gtetrabutylamine hydrobromide are added. The reaction is carried for 16hours at ambient temperature (25° C.) and then 50° C. for 4 hours withgentle stirring. The solution is cooled and the tissue is isolated fromthe reaction mixture. The isolated tissue is washed with PBS and 40%ethanol to remove traces of glycidyl methacrylate and other catalysts.

Example 3 Modification Tissue Using Acrolein

Ten 1 cm by 1 cm bovine pericardium pieces are transferred to 50 mlconical flask containing 10 ml PBS and 0.60 ml acrolein. The tissue isincubated for 6 hours at ambient temperature (25° C.). An additional 0.1g acrolein is added to the mixture at every 1.5 hours up to six hours.Finally, the reaction is continued for 4° C. for 48-120 hours withgentle stirring. The tissue is isolated from the reaction mixture and iswashed with PBS and 40% ethanol to remove unreacted acrolein.

Example 4 Modification Tissue Using Methacrylic Anhydride orMethacryloyl Chloride

Treatment in Aqueous Medium

Ten 1 cm by 1 cm bovine pericardium pieces are transferred to 100 mlround bottom flask containing 10 ml 100 mM sodium borate buffer, pH 9.5and a magnetic stir bar. To this solution, 2 ml methacrylic anhydrideand 2 ml triethylamine are added. The reaction is carried out for 6hours at ambient temperature (25° C.) and then at 0° C. for 72 hourswith gentle stirring. The solution is cooled and the tissue is isolatedfrom the reaction mixture. The isolated tissue is washed with PBS and40% ethanol to remove unreacted methacrylic anhydride.

Treatment in Organic Solvent (Dimethyl Sulfoxide)

Ten 1 cm by 1 cm bovine pericardium pieces are transferred to 100 mlround bottom flask containing 10 ml dimethyl sulfoxide and a magneticstir bar. To this solution, 0.1 ml triethylamine and 0.02 ml methacrylicanhydride are added. The reaction is carried for 24-48 hours at ambienttemperature (30° C.) and then the tissue is isolated from the reactionmixture. The isolated tissue is washed with PBS and 40% ethanol toremove unreacted methacrylic anhydride and stored at 4° C. inrefrigerator until further use. In some cases the tissue is usedimmediately and without ethanol treatment in polymerization andcrosslinking step.

Example 5 Crosslinking of Unsaturated Group Modified Tissue Using FreeRadical Photopolymerization

Two 1 cm by 1 cm pieces of unsaturated group modified bovine pericardiumtissue prepared per Examples 1-4 are incubated for 30 minutes each in20% ethanol, 50% ethanol and 70% ethanol. 50 mg of Irgacure 2959 (freeradical photoinitiator) is dissolved in 1 ml n-vinyl pyrrolidinone toprepare a monomer solution. The 70% ethanol incubated tissue istransferred to Irgacure solution and incubated for 4 h at 25° C. andthen for 12 h at 0° C. The tissue is removed from solution is placed ona glass plate and exposed to a Black-Ray UV lamp (360 nm light, 10000mW/cm2 intensity) for 5 minutes for each side. The crosslinked tissue isremoved and washed with 10 ml PBS to remove unreacted monomer, initiatorfragments and water soluble non-crosslinked polymer. The crosslinkedtissue shows higher shrink temperature as compared to uncrosslinkedtissue indicating crosslinking of the tissue. Presence of polyvinylpyrrolidinone in the tissue is conformed by IR spectrophotometer. Thecrosslinked tissue also showed a different texture as compared touncrosslinked tissue when touched and felt by a human hand. Thecrosslinked tissue also shows resistance to pepsin digestion.

In some embodiments, the tissue is infused with monomer withoutdehydrating using alcohol solutions and the subsequentphotopolymerization treatment showed higher shrink temperatureindicating crosslinking.

Example 6

Crosslinking of Unsaturated Group Modified Tissue Using ThermallyInduced Radical Polymerization

Two 1 cm by 1 cm pieces of unsaturated group modified bovine pericardiumtissue prepared per Examples 1-4 are incubated for 30 minutes each in20% ethanol, 50% ethanol and 70% ethanol. 10 mg ofazobisisobutyronitrile (thermal free radical photoinitiator) isdissolved in 1 ml vinyl pyrrolidinone. The 70% ethanol incubated tissueis transferred to the vinyl pyrrolidinone solution and incubated for 4 hat 25° C. and then for 12 h at 0° C. The tissue is removed, placedbetween two glass slides. The tissue is then heated in an ovenmaintained at 55° C. (below the shrink temperature of uncrosslinkedtissue) for 4 hours. The tissue is washed with 20 ml acetone and 20 mlPBS to remove water/acetone soluble unwanted products.

Example 7 Crosslinking of Unsaturated Group Modified Tissue UsingRadiation or Electron Beam Induced Polymerization

Two 1 cm by 1 cm pieces of unsaturated group modified bovine pericardiumtissue prepared per Example 1-4 are incubated for 30 minutes each in 20%ethanol, 50% ethanol, 70% ethanol and finally in n-vinyl pyrrolidinone.The tissue is removed from vinyl pyrrolidinone and exposed to gammaradiation (total dose 3 Mrad). The exposed tissue is washed with PBS andexamined for crosslinking. No photoinitiator is used to initiate thepolymerization and crosslinking of tissues.

Example 8 Crosslinking of Unsaturated Group Modified Tissue UsingMixture of Hydrophilic and Hydrophobic Monomers

Two 1 cm by 1 cm pieces of unsaturated group modified bovine pericardiumtissue prepared per example 1 are incubated for 30 minutes each in 20%ethanol, 50% ethanol and 70% ethanol. 50 mg of Irgacure 2959 (freeradical photoinitiator) is dissolved in 0.8 ml vinyl pyrrolidinone(hydrophilic monomer) and 0.2 ml octyl methacrylate (hydrophobicmonomer). The 70% ethanol incubated tissue is transferred to Irgacuresolution and incubated for 4 h at 25° C. and then for 12 h at 0° C. Thetissue is removed from solution and then is placed on a glass plate andexposed to Black-Ray UV lamp (360 nm light, 10000 mW/cm2 intensity). Thecrosslinked tissue is washed with large amount of water and 100 percentethanol to remove organic and water soluble impurities. The ratio ofhydrophilic to hydrophobic monomer may be changed to control the watercontent of the crosslinked tissue. Many monomers known in the freeradical polymerization chemistry art that provide hydrophobic andhydrophilic polymers can be used to crosslink the tissue.

Example 9 Crosslinking of Unsaturated Group Modified Tissue UsingFunctional Monomer

Two 1 cm by 1 cm pieces of unsaturated group modified bovine pericardiumtissue prepared per example 1 are incubated for 30 minutes each in 20%ethanol, 50% ethanol and 70% ethanol. 50 mg of 2,2-demethoxy-2-phenylacetophenone (free radical photoinitiator) is dissolved in 0.5 mlglycidyl methacrylate (monomer containing epoxy group) and 0.5 mln-vinyl pyrrolidone. The 70% ethanol incubated tissue is transferred toglycidyl methacrylate solution and incubated for 4 h at 25° C. and thenfor 12 h at 0° C. The tissue is removed from solution is placed on aglass plate and exposed to Black-Ray UV lamp (360 nm light, 10000 mW/cm2intensity) for 15 minutes each side. The crosslinked tissue is washedwith water and 100 percent methanol to remove water and organic solubleimpurities. The epoxy functionality of glycidyl may be further used tointroduce other chemical moieties such as, by way of example, and notlimitation, bioactive compounds or it may, for example, and withoutlimitation, be used to further crosslink the tissue using di- orpolyfunctional amines such as, by way of example, and not limitation,hexamethylene diamine

Example 10 Crosslinking of Unsaturated Group Modified Tissue UsingMonomers that Produce Elastomeric Polymers

Crosslinking Using Poly(Dimethyl Siloxane) Based Monomer

50 mg of 2,2-demethoxy-2-phenyl acetophenone (free radicalphotoinitiator) is dissolved in 0.2 Poly(dimethylsiloxane), vinylterminated (Sigma-Aldrich Product Number: 43,301-2) and 0.8 ml n-vinylpyrrolidone. Two 1 cm by 1 cm pieces of unsaturated group modifiedbovine pericardium tissue prepared per example 1 are incubated for 30minutes each in 20% ethanol, 50% ethanol and 70% ethanol and finally inPoly(dimethylsiloxane) solution prepared as indicated earlier. Thetissue is removed from solution and is placed on a glass plate andexposed to Black-Ray UV lamp (360 nm light, 10000 mW/cm2 intensity) fromboth sides. The crosslinked tissue is washed with water and 100 percentmethanol to remove water and organic soluble impurities. Incorporationof synthetic silicone based rubber can assist to improve mechanicalproperties of the tissue and improve oxygen permeability.

Example 11 Tissue Modified with Thermosensitive Polymer

Crosslinking of Acrylic Modified Tissue Using Monomer that ProducesThermosensitive Polymer/Hydrogel

50 mg of 2,2-demethoxy-2-phenyl acetophenone (free radicalphotoinitiator) is dissolved in 0.4 ml n-vinyl pyrrolidone and 0.6 gn-isopropylacrylamide. Two 1 cm by 1 cm pieces of acrylic acidsuccinimide ester modified or glycidyl methacrylate modified bovinepericardium tissue prepared per example 1 or 37 is incubated for 30minutes each in 20% ethanol, 50% ethanol and 70% ethanol and finallyn-vinyl pyrrolidinone solution. The tissue is then transferred ton-isopropylacrylamide solution in n-vinyl pyrrolidinone (40%) containing1000 ppm Irgacure 2959 and incubated for 4 h. The tissue is removed fromsolution is placed on a glass plate and exposed to Black-Ray UV lamp(360 nm light, 10000 mW/cm2 intensity) for 5 minutes each side. Theexposed tissue is washed with 100% alcohol to remove unreacted monomersand initiator fragments and then incubated in cold PBS solution (4° C.)for 24 hours. The polymerized n-isopropylacrylamide absorbs water at lowtemperature (4° C.) and also permits collagen chains to re-hydrate. Thefully absorbed tissue is incubated in PBS maintained at 37° C. At 37°C., n-isopropylacrylamide turns hydrophobic but collagen chains remainhydrated. Alternatively, a 20-50 percent n-isopropylacrylamide solutionin water may also be used in place of ml n-vinyl pyrrolidone andn-isopropylacrylamide mixture for polymerization and crosslinkingreaction using 500 ppm Irgacure 2959 as photoinitiator.

Example 12 Bioprosthetic Tissue and Polyvinyl Alcohol Composite

Crosslinking of Unsaturated Group Modified Tissue Using Vinyl Acetatewhich is Converted into Polyvinyl Alcohol

Vinyl acetate is vacuum distilled prior to use to remove polymerizationinhibitor. 50 mg of 2,2-demethoxy-2-phenyl acetophenone (free radicalphotoinitiator) is dissolved in 1 ml inhibitor free vinyl acetate. Two 1cm by 1 cm pieces of acrylic acid succinimide ester modified bovinepericardium tissue prepared per example 1 are incubated for 30 minuteseach in 20% ethanol, 50% ethanol and 70% ethanol andn-methylpyrrolidinone. The tissue is then transferred to vinyl acetatesolution and incubated for 4 h. The tissue is removed from solution andthen placed on a glass plate and exposed to Black-Ray UV lamp (360 nmlight, 10000 mW/cm2 intensity) for 15 minutes each side. The exposedtissue is washed with 100% alcohol to remove unreacted monomers andinitiator fragments. The tissue is then incubated in 1% p-toluenesulfonic acid solution in water to hydrolyze polyvinyl acetate. Briefly,the treated tissue is incubated in 10 ml distilled water containing 100mg p-toluene sulfonic acid at 50° C. for 3 days.

Using a similar procedure, acrylonitrile monomer can be can be used inplace of vinyl acetate in tissue crosslinking. The nitrile groups inpolymerized polyacrylonitrile can be partially or completely hydrolyzedunder mild acidic/basic conditions to produce a mechanically strongpolyacrylonitrile-co-polyacrylic acid hydrogel in the tissue matrix.

Example 13 Crosslinked Tissue Complexed with Biologically ActiveCompounds or Synthetic Polymers

Iodine solution is prepared by dissolving elemental iodine (0.1%) andpotassium iodide (0.4%) in 10% ethanol. Vinyl pyrrolidinone crosslinkedtissue (prepared per Example 5 or 37) is incubated with iodine solutionfor 3 hours at 25° C. temperature and is then removed from the solutionand washed with PBS solution. The iodine complexes withpolyvinylpyrrolidinone in the treated tissue. The brown color ofiodine-PVP complex is noticeable on the tissue surface. The iodinecomplexed tissue may be applied on a wound and the iodine released inthe wound area is used to control infection or bacterial growth. Othercompounds that form complex with polyvinyl pyrrolidinone such as, by wayof example, and not limitation, antibiotics may also be used.

Example 14 Tissue and Biodegradable Polymer Composite

Tissue and Biodegradable Polymer Composite Wherein the BiodegradablePolymer is Crosslinked

Crosslinking of Acrylic Modified Tissue Using BiodegradableMultifunctional Macromonomer (Water-Soluble)

Part 1: Synthesis of Polyethylene Glycol Lactate Copolymer (10KL5)

30.0 g of PEG 10000, 4.3 g of dl-lactide and 30 mg of stannous octoateare charged into 100 ml Pyrex pressure sealing tube. The tube is thenconnected to an argon gas line and sealed under argon. The tube is thenimmersed in oil bath maintained at 140° C. The reaction is carried outfor 16 h at 140° C. The polymer from the tube is recovered by breakingthe Pyrex tube. The polymer is then dissolved in 70 ml toluene andprecipitated in 2000 ml cold hexane. The precipitated polymer isrecovered by filtration and dried under vacuum for 1 day at 60° C. It isthen immediately used in the next reaction.

Part 2: End-Capping of 10KL5 with Polymerizable or Crosslinkable Group(10KL5A2)

30 g of 10KL5 is dissolved in 450 ml dry toluene. About 50 ml of tolueneis distilled out to remove traces of water from the reaction mixture.The warm solution is cooled to 65° C. To this warm solution, 1.6 g oftriethyl amine and 1.5 g acryloyl chloride are added. The reactionmixture is then stirred for 30 minutes at 50-60° C. and filtered. Thereactive precursor is precipitated by adding the filtrate to 2000 mlcold hexane. The precipitated polymer is recovered by filtration. It isthen dried under vacuum for 12 h at 50° C.

Part 3: Crosslinking of Acrylic Modified Tissue Using 10KL5A2

50 mg of 2,2-demethoxy-2-phenyl acetophenone (free radicalphotoinitiator) is dissolved in 0.9 ml n-vinyl pyrrolidone (NVP) and 0.1g 10KL5A2. Two 1 cm by 1 cm pieces of acrylic acid succinimide estermodified bovine pericardium tissue prepared per examples 1 to 4 isincubated for 30 minutes each in 20% ethanol, 50% ethanol and 70%ethanol and n-vinyl pyrrolidinone. The tissue is then transferred to10KL5A2 solution and incubated for 4 h. The tissue is removed fromsolution is placed on a glass plate and exposed to Black-Ray UV lamp(360 nm light, 10000 mW/cm2 intensity) for 15 minutes each side. Theexposed tissue is washed with 100% alcohol to remove unreacted monomersand initiator fragments and then incubated in cold PBS solution (4° C.)for 24 hours. The lactate ester bonds in the polymerized 10KL5A2hydrolyze in presence of water and make the tissue susceptible forenzymatic degradation. The ratio of NVP to 10KL5A2 could be changed tocontrol biodegradation, swelling and other properties of thetissue/polymer composite matrix.

In another variation of this procedure, a bioactive compound such as, byway of example, and not limitation, heparin may be added in the monomermixture prior to photopolymerization. The bioactive compound is releasedas 10KL5A2 undergoes hydrolysis. In another variation of this method, 5ml of 20% solution of 10KL5A2 in PBS containing 400 ppm Irgacure 2959 isused to suspend 1 million human fibroblast cells. The unsaturated groupmodified tissue is incubated for 30 minutes in the polymer solution. Thesolution is taken out and about 0.2 ml solution is sprayed on the tissuesurface. The solution on the surface and tissue are exposed to 2 minuteswith Black-Ray UV lamp (360 nm light, 10000 mW/cm2 intensity). Thepolymerized 10KL5A2 with encapsulated cells is clearly seen on thetissue surface. Most of the cells remained viable and tolerate theencapsulation process.

Example 15 Tissue and Biodegradable Polymer Composite

Tissue and Biodegradable Polymer Composite Wherein the BiodegradablePolymer is Hydrophobic

Crosslinking of Acrylic Modified Tissue Using BiodegradableMultifunctional Macromonomer (Water-Insoluble or Substantially WaterInsoluble)

Part 1: Preparation of Trifunctional Lactate Liquid Copolymer (TMPTL).

Trimethylol propane triol (TMPT) is dried under vacuum at 60° C. for 16hours. 2 g of dry TMPT, 17.5 g of dl-lactide, and 20 mg of stannousoctoate are charged into a 3 necked flask equipped with Teflon coatedmagnetic stirring needle and nitrogen inlet. The flask is then immersedin a silicone oil bath maintained at 160° C. The reaction is carried outfor 5 h under nitrogen atmosphere. The reaction mixture is then cooledto room temperature. The mixture is then dissolved in 10 ml toluene. Thehydroxyl-terminated liquid lactate polymer is isolated by pouring thetoluene solution in large excess cold hexane. It is further purified byrepeated dissolution-precipitation process from toluene-hexanesolvent-nonsolvent system and dried under vacuum at 60° C. It is thenimmediately used for acrylate end capping reaction described below.

Part 2: End Capping of Trifunctional Polylactide Polymer with AcrylateGroup (TMPTLA).

10 g of TMPT initiated lactate synthesized previously is dissolved in150 ml dry benzene. From this solution 20 ml benzene is distilled out toremove unwanted moisture from the reaction mixture. The solution iscooled to 0° C. in ice bath. 2.7 ml of triethyl amine and 1.8 mlacryloyl chloride are added dropwise to the cold lactate solution. Themixture is refluxed under nitrogen atmosphere for 3 h. The solution isfiltered to remove triethylamine hydrochloride. The acrylate ester isthen isolated by pouring the filtered solution in large excess coldhexane. It is further purified by repeated (3 times) precipitation fromtoluene-cold hexane system. The liquid polymer is dried under vacuum at40° C. It is stored in an amber-colored bottle under nitrogenatmosphere.

Part 3: Crosslinking of Acrylic Modified Tissue Using TMPTLA.

50 mg of 2,2-demethoxy-2-phenyl acetophenone (free radicalphotoinitiator) is dissolved in 0.9 ml n-vinyl pyrrolidone and 0.1 gTMPTLA. Two 1 cm by 1 cm pieces of acrylic acid succinimide estermodified bovine pericardium tissue prepared per example 1 are incubatedfor 30 minutes each in 20% ethanol, 50% ethanol and 70% ethanol andn-vinyl pyrrolidinone. The tissue is then transferred to TMPTALAsolution and incubated for 4 h. The tissue is removed from the solutionis placed on a glass plate and exposed to Black-Ray UV lamp (360 nmlight, 10000 mW/cm2 intensity) for 5 minutes each side. The exposedtissue is washed with 100% alcohol to remove unreacted monomers andinitiator fragments and then incubated in cold PBS solution (4° C.) for24 hours. The lactate ester bonds in the polymerized TMPTALA hydrolyzein presence of water and make the tissue susceptible for enzymaticdegradation.

Example 16 Tissue-Synthetic Biodegradable Crosslinkable PolymerComposite

Five 1 cm by 1 cm pieces of acrylic acid succinimide ester modifiedbovine pericardium tissue are cryogenically ground (at liquid nitrogentemperature) to make a tissue powder. 50 mg of 2,2-demethoxy-2-phenylacetophenone (free radical photoinitiator) is dissolved 0.1 ml n-vinylpyrrolidone and 0.9 g TMPTLA. 1 g of tissue powder is mixed with 200 mgTMPTLA monomer and 1 g sodium chloride (to induce porosity) and themixture poured into a mold cavity. The shape of the cavity is similar toshape of human meniscus tissue. The mixture in the cavity is exposed toBlack-Ray UV lamp (360 nm light, 10000 mW/cm2 intensity) for 15 minutesto cure the TMPTLA polymer. The cured product is washed with 100%ethanol to remove unreacted monomer and then with water to remove sodiumchloride (to induce porosity). The cured tissue/synthetic polymer can beused as a biodegradable meniscus implant or a scaffold for tissueengineering.

Collagen protein powder modified with unsaturated groups per Examples1-4 may also be used in place of tissue powder.

Example 17 Crosslinking of Unsaturated Group Modified Tissue Using Di orPolyfunctional Mercapto Compounds

In this crosslinking method, mercapto group from the crosslinker reactwith unsaturated group in the modified tissue (Examples 1-4) and producea crosslinked tissue.

In a 50 ml polypropylene tube, 7.5 ml 50 mM sodium bicarbonate buffer(pH 9-12) and 2.5 ml acetonitrile are mixed. Five 1 cm by 1 cm pieces ofacrylic acid succinimide ester modified bovine pericardium tissue(Examples 1-3) are suspended in the acetonitrile solution. 0.2 mltrimethylolpropane tris(3-mercaptopropionate) (Sigma-Aldrich ProductNumber: 38,148-9) is added to the tube and the mixture is vortexed for60 minutes. The reaction is continued for 6 h at room temperature and 12hours at 4° C. The tissue is separated from the mixture, washed with PB5 and 100% ethanol and then finally stored in 100% ethanol until use.

Example 18 Crosslinking of Tissue with Biodegradable Crosslinker

Crosslinking of Tissue Using Non-Polymeric Degradable Crosslinker

Synthesis of a Biodegradable Crosslinker; Hydroxylamine Succinate NHSEster

Part 1: Hydroxylamine Succinate

To a solution of 2 g hydroxy amine in 100 ml dry benzene and 100 mlpyridine, 9.4 g succinic anhydride are added and the reaction mixturestirred at room temperature for 2 h and then refluxed for 2 h. Thesolvent is evaporated under vacuum and the residue is purified by flashchromatography using silica gel.

Part 2: Hydroxylamine Succinate NHS Ester.

Part 2: To a cold (4° C.) solution of 5 g Hydroxylamine Succinate and5.1 g n-hydroxysuccinimide in 120 DMF, 11.8 g 1,3-dicyclohexylcarbodiimide in 30 ml of DMF and is added under nitrogen atmosphere. Thereaction is continued at room temperature for 8 h and urea precipitateis filtered. The filtrate is evaporated and the crude compound isrecovered. The compound is further purified by flash chromatography.

Part 3: Crosslinking of Pericardial Tissue with Hydroxylamine SuccinateNHS Ester.

10 pieces of 1 cm by 1 cm bovine pericardium pierces are transferred to50 ml polypropylene centrifuge tube containing 10 ml PBS. 250 mgHydroxylamine Succinate NHS ester in 0.5 ml dimethyl sulfoxide is addedto the tube and the solution is vortexed for 15 minutes. Themodification reaction is carried for 6 hours at ambient temperature (25°C.) and then for 12 hours at 4° C. with gentle shaking. The reaction isterminated by washing the tissue with 20 ml distilled water 3 times.Finally, the tissue is lyophilized and stored at −20° C. until use.

The ester linkage hydroxylamine succinate in the crosslinker ishydrolyzed when exposed to physiological conditions such PBS, pH 7.2.The hydrolyzed tissue degrades by normal enzymatic degradation.

Example 19 Crosslinking Using Polymeric Degradable Crosslinker

10 pieces of 1 cm by 1 cm bovine pericardium tissue are transferred to50 ml polypropylene centrifuge tube containing 10 ml PBS. 1.0 g 4arm-n-hydroxysuccinimide ester of polyethylene glycolcarboxymethylene-butyric acid, average molecular weight 10000 Daltons(obtained from Shearwater Polymers, 4 arm, product CM-HBA-NS-10K) isadded to the tube and the mixture is vortexed for 5 minutes. The tissueis isolated from the tube, washed with distilled water and used as abiodegradable matrix for various medical and tissue engineeringapplications. Upon implantation, the glutarate ester bond inCM-HBA-NS-10K undergoes hydrolysis. After hydrolysis of the crosslinker,tissue degrades by a normal enzymatic pathway. Additional crosslinkingagents such as crosslinking with EDC may also be used to control tissueproperties or degradation time.

Example 20 Bioprosthetic Tissue Incorporated with CrosslinkedBiodegradable Hydrogel

Part 1: Synthesis of Polyethylene Glycol Lactate Copolymer (PEG20KL5).

50 g PEG molecular weight 20000 is dried at 120° C. under vacuum for 10hours. 10.0 g dry PEG molecular weight 20000, 2.2 g of dl-lactide and100 mg of stannous octoate are charged into 100 ml flame dried roundbottom flask. The flask is then connected to an argon gas line and thenimmersed in an oil bath maintained at 160° C. The polymerizationreaction is carried out for 16 h at 160° C. The polymer is thendissolved in 100 ml toluene and precipitated in 2000 ml cold hexane. Theprecipitated polymer is recovered by filtration and dried under vacuumfor 1 day at 60° C. It then immediately used in next step.

Part 2: End-Capping of PEG20KL5 with Polymerizable or CrosslinkableGroup (PEG20KL5A).

30 g of PEG20KL5 is dissolved in 450 ml dry toluene. About 50 ml oftoluene is distilled out to remove traces of water from the reactionmixture. The solution is cooled to 65° C. To this warm solution, 0.6 gof triethyl amine and 0.5 g acryloyl chloride are added. The reactionmixture is then stirred for 30 minutes at 50-60° C. and filtered. Thereactive crosslinkable precursor is precipitated by adding the filtrateto 2000 ml cold hexane. The precipitated polymer is recovered byfiltration. It is then dried under vacuum for 12 h at 50° C.

Part 3: Incorporation of Hydrophilic Crosslinkable Biodegradable Polymerin the Pericardial Tissue.

A 3 cm×3 cm piece is cut from a cleaned uncrosslinked bovine pericardialsac. The tissue is then incubated for 30 minutes each in 20% ethanol,40% ethanol, 80% ethanol and finally in 100% ethanol. In a 500 mlbeaker, 10 g PEG20KL5A, 0.5 g Chlorhexidene Gluconate, 0.050 g Irgacure2959 are dissolved in 90 g methanol. The 100% ethanol-treated tissue istransferred into Chlorhexidene Gluconate solution and is incubated for 2hours. The tissue is removed from the solution and exposed to long UVlight emitting at 360 nm for 5 minutes (Black-Ray light source, model3-100A, Flood 365 nm, intensity 30 mW/cm2).

Example 21 Bioprosthetic Tissue-Hydrophilic Non-CrosslinkedBiodegradable Polymer Composite

Biodegradable Tissue Based Patch for Controlled Drug Delivery

Part 1: Synthesis of Polyethylene Glycol Lactate Copolymer (PEG8K50)

50 g PEG molecular weight 8000 (Carbowax 8000) is dried at 120° C. undervacuum for 10 hours. 10.0 g dry PEG molecular weight 8000, 18.0 g ofdl-lactide and 100 mg of stannous octoate are charged into 100 ml flamedried round bottom flask. The flask is then connected to an argon gasline and then immersed in an oil bath maintained at 160° C. Thepolymerization reaction is carried out for 16 h at 160° C. The polymeris then dissolved in 100 ml toluene and precipitated in 2000 ml coldhexane. The precipitated polymer is recovered by filtration and driedunder vacuum for 1 day at 60° C.

Part 2: Incorporation of Hydrophilic Non-Crosslinked BiodegradablePolymer (PEG8K50) in the Pericardial Tissue.

A 3 cm×3 cm piece is cut from a cleaned uncrosslinked bovine pericardialsac. The tissue is incubated for 30 minutes each in 20% ethanol, 40%ethanol, 80% ethanol and finally in 100% ethanol. In a 500 ml beaker, 10g polyethylene glycol-lactate copolymer (PEG8K50), and 1 g rifampin aredissolved in 90 g chloroform. The 100% ethanol-treated tissue istransferred into rifampin solution and is incubated for 2 hours. Thetissue is removed from the solution and the solvent is evaporated by airdrying. The reddish yellow rifampin-PEG-lactate coating is clearlyvisible to the naked eye. The dry tissue is sterilized using ethyleneoxide and used as a biodegradable drug delivery patch. The polyethyleneglycol-lactate polymer acts as a hydrophilic non-crosslinkedbiodegradable polymeric drug delivery matrix for Rifampin.

The tissue may be mechanically resurfaced or perforated to improve theadhesion of polymer to the tissue.

The same procedure may also be used to incorporate biodegradable polymerin glutaraldehyde crosslinked tissue or EDC crosslinked tissue.

Example 22 Incorporation of Hydrophobic Non-Crosslinked SyntheticBiodegradable Polymer in the Pericardial Tissue

Biodegradable Tissue Based Patch for Controlled Drug Delivery

Seven 1 cm×1 cm pieces are cut from a cleaned uncrosslinked bovinepericardial sac. The tissue is then incubated for 30 minutes each in 20%ethanol, 40% ethanol, 80% ethanol and finally in 100% ethanol and dried.

In a 50 ml beaker, 200 mg polylactide-co-polyglycolide (50:50),molecular weight 50000 (Sigma-Aldrich Catalog number 43,044-7) isdissolved in 3 ml chloroform. 20 mg rifampin is added to the polymersolution. The 100% ethanol treated tissue is transferred into rifampinsolution and is incubated for 2 hours. The tissue is removed from thesolution and the chloroform is evaporated by air drying. The reddishyellow rifampin-polymer coating is clearly visible to the naked eye. Ifnecessary, vacuum drying may, for example, and without limitation, beused to completely remove the chloroform. Finally the tissue issterilized using ethylene oxide.

Two pieces of rifampin-treated tissue are incubated in 2 ml PBS at 37°C. and the release of rifampin is monitored over a period of 72 hours.The PBS is exchanged at 10 minutes, 6 hours, 24 hours, 48 hours and 72hours time intervals. All incubated solutions had light yellow colorvisible to the naked eye indicating the presence of rifampin in thesolution. The concentration of rifampin in the eluted solution isanalyzed by UV-VIS spectrophotometry.

Using a similar method, a high molecular weight polydl-lactide,molecular weight 100000 (Sigma-Aldrich Catalog Number 53,116-2) is usedin place of polylactide-co-polyglycolide (50:50) to form atissue-biodegradable polymer composite. This polymer takes a much longertime to degrade and shows a different rifampin release profile.

The biodegradable polymer used could be liquid or low melting solid.Using similar methods to those described above, a non-polymeric drugcarrier such as, by way of example, and not limitation, sucrose acetateor vitamin E or vitamin E acetate may be also used as a carrier andincorporated in the tissue. If a biostable tissue is matrix is desired,then a crosslinked tissue such as, by way of example, and notlimitation, glutaraldehyde crosslinked tissue may, for example, andwithout limitation, be used in place of non-crosslinked tissue.

Example 23 Porcine Pericardial Patch Coated with Biodegradable Polymer(Synthetic Biodegradable Hydrogel)

From a freshly obtained piece of porcine pericardial sac, A 3 cm×3 cmpiece of tissue is cut. This tissue is incubated in 1% Eosin Y solutionin PBS for 10 minutes. The tissue is rinsed with fresh PBS to removeexcess of eosin solution from the tissue and used in the coatingprocess.

20 g 10KL5A2 (Example 14), 80 g saline solution buffered with 1000 mMtriethanol amine buffer (pH 7.4) and 1 ml vinyl pyrrolidinone are mixedin 1000 ml beaker. Eosin stained pericardial tissue is placed inside thesolution. Care is taken to completely cover the tissue surface with10KL5A2 solution. The tissue is then exposed to 514 nm light either fromargon laser (American Argon ion laser, Model 905 emitting at 532 nm, 100mW/cm2) or xenon lamp (intensity 0.5 W/cm2). Theeosin-triethanolamine-10KL5A2-light initiates a photopolymerizationreaction at the tissue interface forming a thin hydrogel film on thetissue surface. The thickness of the film is dependent on the exposuretime, monomer concentration, eosin concentration and light intensity. Ato 2000 micron coating is obtained. The biodegradable hydrogel coatedtissue can be used as a coated tissue patch. If necessary, multipleeosin and monomer solution treatments may be performed to achieve adesirable thickness.

Using a similar approach to that described above, an albumin modifiedwith polymerizable groups may, for example, and without limitation, beused to coat tissue surface. The albumin may be bovine albumin or humanalbumin. The human albumin may be obtained by recombinant technology.

A crosslinked tissue such as, by way of example, and not limitation,glutaraldehyde crosslinked tissue may, for example, and withoutlimitation, be used in place of uncrosslinked tissue if a biostablematerial is desired.

Example 24 Porcine Pericardial Patch Coated with Synthetic BiodegradablePolymer Hydrogel Formed by Condensation Polymerization

An ethylene oxide sterilized air assisted sprayer is used in conjunctionwith aqueous solutions of polymerizable monomers. Solution consisted ofa 14.4% solution of 4 arm-n-hydroxysuccinimide ester of polyethyleneglycol carboxymethylene-butyric acid, average molecular weight 10000Daltons (Shearwater 4 arm CM-HBA-NS-10K) is dissolved in 0.01M phosphatebuffer at pH 4.0 and is sterile-filtered (Pall Gelman syringe filter,p/n 4905) and drawn up in a sterile 5 cc syringe. Solution 2 consistedof a 1.2% solution of a dilysine (purchased from Sigma Chemicals)dissolved in 0.1M borate buffer at pH 11 with 0.5 mg/mL methylene bluefor visualization and is also sterile-filtered and drawn up in a sterile5 cc syringe. These solutions, when combined 1:1 on a volumetric basis,result in a 1:1 ratio of NHS ester to amine end group. The final %solids after combination is 7.5%. The two syringes are individuallyloaded in the two separate receptacles through a luer-lok type oflinkage. Airflow from a regulated source of compressed air (an aircompressor such as, by way of example, and not limitation, thosecommercially available for airbrushes) is connected to the device usinga piece of Tygon tube. On compressing the syringe plungers a steadyspray of the two liquid components is observed. A 10 cm×5 cm piece iscut from a cleaned uncrosslinked bovine pericardial sac or AlloDerm®tissue marketed by LifeCell Corporation, NJ. The spray from the syringeis directed to the piece of pericardial tissue, and a hydrogel coatingis formed on the surface of the tissue. Alternatively, the coatingcompositions and spray system can be purchased as DuraSeal fromConfluent Surgical Inc., MA and used. Within a short period of time(less than a minute) an area of 10 cm×5 cm could be coated with ease.The coating can be applied on both the sides of the tissue. Thepolyethylene glycol coating makes the tissue surface non-cell adhesive.The hydrogel coating is rinsed with saline (the hydrogel coating isresistant to rinsing) and is observed to be well adherent to the tissuesurface. The hydrogel coating can be used to modify biologicalproperties of the tissue. If the coating is applied on one side only,then one surface (coated surface) becomes non-cell adhesive while theuncoated surface remains as a cell adhesive surface. The coating canalso be used to deliver drugs in a controlled manner. This is achievedby adding drug or drug encapsulated microspheres in a coatingformulation before coating the tissue or by incubating the coated patchin drug solution in organic solvent or water and then removing thesolvent. The coated tissue can be used as a barrier for reducingsurgical adhesions. Such a non-cell adhesive tissue patch can be used toreduce post-operative adhesions. The coated tissue loaded with BMPproteins could be used to generate a bone tissue.

A crosslinked tissue such as, by way of example, and not limitation,glutaraldehyde crosslinked tissue may, for example, and withoutlimitation, be used on place of uncrosslinked tissue if a biostablematerial is desired.

Example 25 Prevention of Post-Operative Adhesions Using Coated TissuePatch or Composite Tissue Patch

Rat Cecum Model

Fourteen Sprague Dawley rats with average weight around 260 g aredivided into 2 groups, 7 animals in each group. After following thestandard procedures for administrating anesthesia, a midline incision ismade on the abdomen and a cecum is located in the abdominal cavity.Using a standard surgical cotton gauze pad, an injury is made to theCecum surface by abrading the surface of cecum. The approximate area ofinjury is maintained around 1-2 sq. cm. The injury elicits some bleedingfrom the injured surface. A porcine pericardial tissue-coated with PEGbased hydrogel or DuraSeal based hydrogel is wrapped around the injuredsurface (Examples 20-24, preferably example 24). Composite patch madeusing polytetrafluoroethylene (PTFE) membrane and membrane tissuewherein PTFE membrane serves as non-cell adhesive layer and tissue serveas cell adhesive layer may also be used. The coated tissue is wrappedaround the injured area such that non-adhesive surface is facing to theinjured surface and immobilized by suturing it either to itself or bysuturing it to abdominal wall. Care is taken to ensure that a hydrogelsurface or PTFE in place of a composite is completely covering theinjured cecum surface. 7 animals are treated with coated tissue and 7animals are used as controls which did not receive any tissue composite.The animals are closed after the treatment using sutures and staples,topical antibiotic is applied and the animals are subjected to standarddiet and care as recommended by National Institute of Health. After 14days the rats are subjected to CO2 asphyxiation.

The incisions are reopened and the cecum is observed for the adhesionsin the area of injury. The adhesions are scored as the percent injuredarea involved in adhesion formation with the surrounding organs andperitoneal wall. The results of treated and control animals are analyzedusing standard statistical methods (t-test).

Coated or composite tissue patch prepared as above may be used in herniasurgery to reduce complications due to surgical adhesions.

Example 26 Chemical Modification of Tissue without Crosslinking

Modification of Uncrosslinked Tissue Using Monomethoxy PolyethyleneGlycol Derivatives

Part 1: Synthesis of Monomethoxy Polyethylene Glycol Succinic Ester.Conversion of PEG Hydroxyl Groups into Carboxylic Groups.

10 g monomethoxy polyethylene glycol, molecular weight 400 (PEG-400M) isdried at 60° C. overnight under vacuum prior to use. 10 g PEG-400M isdissolved in 35 ml dry pyridine and 35 ml benzene. 3.5 g succinicanhydride or 4.0 g of glutaric anhydride is added to it and the solutionis refluxed for 2 h under nitrogen atmosphere. Most of the pyridine isdistilled out and the polymer is isolated by pouring the coldconcentrated pyridine solution to cold 4000 ml hexane and dried undervacuum at 60° C. and used immediately in a subsequent carboxyl groupactivation reaction.

Part 2: Activation of Acid Group Using n-Hydroxysuccinimide(PEG-400MNHS).

To a solution of 10 g of PEG-400M succinate or glutarate in 100 ml drymethylene chloride are added 2.5 g n-hydroxysuccinimide and 5.9 g1,3-dicyclohexyl carbodiimide. The reaction mixture is cooled to 0° C.using ice bath and stirred overnight under nitrogen atmosphere.Dicyclohexylurea is removed by filtration. The filtrate is evaporatedand the residue obtained is redissolved in 10 ml toluene. The toluenesolution is precipitated in 2000 ml cold hexane.

Alternatively, monomethoxy PEG NHS derivates may be purchased fromSigma-Aldrich or Sherewater Inc.

Part 3: Modification of Tissue Using Monomethoxy Polyethylene GlycolSuccinic Ester (PEG-400MNHS)

Porcine pericardial sac is obtained from a local abbotair and is cleanedto remove blood and fatty tissue from the surface. Ten 1 cm by 1 cmpieces are cut from the cleaned pericardial sac and transferred to 10 ml20 mM phosphate buffer solution (PBS, pH 7.2).

200 mg monomethoxy polyethylene glycol succinic ester or glutaric ester(PEG-400MNHS, part 2) is added to the tissue/PBS mixture. The solutionis vortexed for 5 minutes and tissue is incubated at room temperaturefor 5 minutes to 12 hours, preferably 6 hours. The tissue is separatedfrom the crosslinker mixture and washed with 20 ml PBS solution 3 timesto remove unreacted chemicals. The PEG-modified tissue shows does notshow significant increase in shrink temperature and the modified tissuedegrades when exposed to collagenease or pepsin solution for prolongedperiods of time indicating its susceptibility to enzymatic degradation.

The tissue treatment can be done just prior to surgical implantation ifthe PEG derivative, tissue and PBS is provided as a kit.

Example 27 Chemical Modification of Tissue without Crosslinking

Modification of Uncrosslinked Tissue Using Anhydrides

Porcine pericardial sac is obtained from a local abbotair and is cleanedto remove blood and fatty tissue from the surface. Ten 1 cm by 1 cmpieces are cut from the cleaned pericardial sac and transferred to 10 ml20 mM sodium borate buffer (pH 9.5). 0.5 g succinic anhydride is addedto the tube. The solution is vortexed for 5 minutes and tissue isincubated at room temperature for 12 hours. The tissue is separated,washed and stored until use.

Using a similar reaction scheme, the tissue is modified using 0.5 gacetic anhydride or 0.5 g glutaric anhydride. Briefly, two 1 cm by 1 cmpieces of bovine pericardium tissue are incubated for 30 minutes each in20% ethanol, 50% ethanol and 70% ethanol and n-methyl pyrrolidinone. Thetissue is then transferred to a 50 ml flask containing 5 ml n-methylpyrrolidinone, 0.5 g acetic anhydride and 0.5 g triethyl amine. Thetissue is incubated for 6 h at 40° C. and then removed. It is washedwith 20 ml 100% ethanol and 20 ml PBS.

Example 28 Chemical Modification of Tissue without Crosslinking

Modification of Uncrosslinked Porcine Pericardial Tissue Using ActivatedAcid (Acetic Acid n-Hydroxy Succinimide Ester)

Porcine pericardial sac is obtained from a local abbotair and is cleanedto remove blood and fatty tissue contaminants from the tissue surface.Ten 1 cm by 1 cm pieces are cut from the cleaned pericardial sac andtransferred to 10 ml 0.20 M 2-(N-morpholino)ethanesulfonic acid (MES)buffered solution (pH 6.5). 0.5 g Acetic acid n-hydroxy succinimideester) AANHS is added to the buffered solution and the reaction isvortexed for 15 minutes and continued for 6 hours at ambienttemperature. An additional 0.1 g AANHS ester is added to the mixture atevery 1.5 hours. After the reaction, the tissue is isolated and washedseveral times with PBS and finally stored in 50% ethanol solution. Thetreated tissue is sterilized using ethylene oxide.

The modified tissue is degradable upon implantation. The tissue becomesless inflammatory due to chemical modification.

The tissue treatment can be done just prior to surgical implantation ifthe acetic acid n-hydroxy succinimide ester, tissue and PBS is providedas a kit.

Example 29 Shape Preserving Tissue Fixation Methods and Devices

a) Preparation of Helical Coil Shaped Tissue (Tissue Stent)

Untreated long bovine pericardium (BP) tissue strips (approximately 10to 20 cm long and 2-4 mm wide) are transferred to a 500 ml flaskcontaining 100 ml PBS, 12.5 ml triethylamine (TEA) and 12.5 ml glycidylmethacrylate (GM). The solution was heated to 50° C. with constantstirring using magnetic stir bar and held there for 8 hours. The tissuestrips are removed from the solution and washed with PBS, distilledwater, & incubated in 70% isopropanol solution in water for 30 minutes.Finally, strips are then transferred to a tetraethyleneglycoldimethacrylate (TEGDM) monomer containing 500 ppm 2,2-dimethoxy 2-phenylacetophenone (photoinitiator) and incubated at room temperature (30° C.)for one hour. One TEGDM infused strip is helically or spirally wound ona 6 mm diameter glass rod. Both ends of the strip are tied with a threadto prevent unwinding. The helically wrapped monomer infused tissue stripwas then exposed to 360 nm light (Black-Ray UV lamp, 360 nm flood light,10000 mW/cm2 intensity at a distance of 10 cm) for 10 minutes whilegently rotating to expose the tissue from all sides. The coiled tissuewas removed from the glass rod and was washed with PBS and 70%isopropanol to remove unreacted monomer and initiator fragments. Thetissue preserved the helical shape after removal of glass rod mandrelsupport.

b) Preparation of Compressible or Wrinkled Tube from Membrane LikeTissue.

Preparation of Compressible Vascular Graft from Flat Tissue Such as, byWay of Example, and not Limitation, Pericardial Tissue or SubmucosaTissue

Untreated long bovine pericardium (BP) tissue strips (approximately 18to 20 cm long and 15-18 mm wide) are transferred to a 500 ml flaskcontaining 250 ml PBS, 32 ml triethylamine (TEA) and 32 ml glycidylmethacrylate (GM). The solution was heated to 50° C. with constantstirring using magnetic stir bar and held there for 8 hours. The tissuestrips are removed from the solution and washed with PBS, distilledwater, & incubated in 70% isopropanol solution in water for 30 minutes.Finally, strips are then transferred to a 50% n-vinyl pyrrolidinone(NVP) solution in water containing 500 ppm 2,2-dimethoxy 2-phenylacetophenone (photoinitiator) and incubated at room temperature (30° C.)for 1 hour. The NVP infused tissue strip is wrapped on a 6 mm diameterstainless steep screw having threads. The tissue was compressed in sucha manner that it took shape of a threaded region of the screw. Both endsof the strip are tied with thread to prevent unwinding. Thehelically-wrapped monomer-infused tissue strip was then exposed to 360nm light (Black-Ray UV lamp, 360 nm flood light, 10000 mW/cm2 intensityat a distance of 10 cm) for 10 minutes while gently rotating to exposethe tissue from all sides. The tissue was removed from the rod and waswashed with PBS and 70% isopropanol to remove unreacted monomer andinitiator fragments. The tissue duplicated the shape of the threadedscrew. This tissue was sewn to make 5 mm diameter tube. The tubulartissue was compressible similar to commercially available polyesterbased vascular grafts.

In another approach, a pericardial tissue patch modified with glycidylmethacrylate (see example 37 described below) and infused withtetraethyleneglycol dimethacrylate (TEGDM) monomer containing 500 ppm2,2-dimethoxy 2-phenyl acetophenone (photoinitiator) was converted intoa 6 mm diameter 10 cm long tube. The tube was mounted on a 6 mm diameterstainless steel mandrel/rod. The mounted tube was compressed along theaxis of the tube to reduce the its length from 10 cm to 7 cm (30%compression). The tube has several wrinkles uniformly distributed alongthe axis of the tube. The compressed tube was then exposed to UV light(Black-Ray UV lamp, 360 nm flood light, 10000 mW/cm2 intensity at adistance of 10 cm) for 10 minutes while rotating the tube. Thecrosslinked tube was removed from the mandrel and washed with 70%ethanol and then PBS. The tube maintained its compressed shapeindicating shape preservation. The compression also made the tissue morecompliant along the axis of the tube. Such compliant pericardial graftmade from flat tissue such as, by way of example, and not limitation,pericardial tissue may be useful as peripheral, coronary and AV vasculargraft or may be used to make stent graft. The tissue surface is expectedto offer superior hemocompatibility or patency.

Example 30 Bioprosthetic Tissue Coated with Polyethylene GlycolCrosslinked with Gamma Radiation

A 3 cm×3 cm piece is cut from a cleaned uncrosslinked bovine pericardialsac. The tissue is fixed by incubating in 0.2% glutaraldehyde solutionfor 7 days. The tissue is washed with PBS and then incubated for 30minutes each in 20% ethanol, 40% ethanol, 80% ethanol, and finally in100% ethanol. In a 500 ml beaker, 1 g polyethylene oxide molecularweight 100000 Daltons is dissolved in 20 ml chloroform. Theethanol-treated tissue is transferred into polyethylene oxide solutionand incubated for 4 hours. The chloroform is removed by air drying. Thepolyethylene oxide-tissue composite is exposed to gamma radiation (Dose0.1 to 4 Mrad) to crosslink polyethylene oxide. The crosslinkedpolyethylene glycol-tissue composite is incubated in PBS to rehydratethe collagen and polyethylene oxide polymer. The crosslinkedpolyethylene oxide and tissue composite patch is used as a wounddressing.

Example 31 Membrane Like Tissue Loaded with Drugs and Used as a Patchfor Local Delivery

Bioprosthetic Tissue Incorporated with Water Insoluble Drugs

Porcine pericardial sac is obtained from a local abbotair and is cleanedto remove blood and fatty tissue from the surface. Ten 1 cm by 1 cmpieces are cut from the cleaned pericardial sac and then incubated for30 minutes each in 20% ethanol, 50% ethanol and 70% ethanol, and finally100 percent ethanol. The ethanol-treated tissue is then transferred tochlorhexidene acetate solution (30 percent solution in methanol) andincubated for 5 hours. The methanol is evaporated and the drug crystalsare formed throughout the tissue matrix. When such tissue is implantedin an animal, it releases the drug in a controlled manner, possibly as aresult of slow dissolution of drug crystals. A Teflon®-based releaseliner is applied on the tissue surfaces to reduce loss of drug crystalsduring routine handling, transportation and storage. The release lineris removed just prior to implantation.

In another variation of this approach, the tissue is first exposed tosilver nitrate solution followed by exposing to sodium acetate solution.The exposure of silver nitrate solution inside the tissue matrix tosodium acetate solution forms silver acetate ‘in situ’ inside the tissuematrix. The deposited silver salts release silver ions and provideantimicrobial properties to the tissue. The silver salt deposition maybe carried out in the operating room prior to implantation if silversalt, implantable tissue and appropriate salt solution such as, by wayof example, and not limitation, sodium acetate, sodium chloride, sodiumlactate are provided in a sterile manner and as a kit. The tissue usedmay be crosslinked (biostable) or uncrosslinked (biodegradable). Thesame method also could be used to deposit silver salts in collagen basedproducts such as, by way of example, and not limitation, woulddressings, collagen sponge products used for implantation etc. Inanother example, hydroxyapatite may be deposited or formed in the tissuematrix to make a hydroxyapatite-tissue composite matrix. Thehydroxyapatite is added to the tissue matrix using methods known in theorthopaedic biomaterials art. In one embodiment, hydroxy apatite issynthesized inside the tissue matrix by combing calcium ion andphosphate ion solution in presence of tissue. The calcium nitrate[Ca(NO3)2, 4H2O, Aldrich], may be used as calcium source and ammoniumhydrogen phosphate [(NH3)2HPO4, Aldrich] may be used as phosphorussource. The calcium nitrate solution is added to ammonium hydrogenphosphate solution at 40° C. while maintaining the pH using ammoniumhydroxide to 10 and 10.6 to form hydroxyapatite crystals. The molarratio of The Ca and P precursors is maintained 1.6 to makehydroxyapatite crystals. This reaction is carried in presence of tissuesuch as pericardial tissue so that hydroxyapatite is generated in sidethe tissue. The tissue-hydroxyapatite can potentially be used as amatrix for bone formation. If needed bone promoting bioactive compoundssuch as BMPs may be added in the composite matrix to accelerate the boneformation.

Example 32 Crosslinking of Tissues Using Unsaturated Acids

32a-1) Synthesis of Activated Unsaturated-Acids (n-HydroxysuccinimideEster)

Synthesis of Fumaric Acid n-Hydroxysccinimide Ester (FUNHS) Using AcidChloride Route.

8.2 g N-hydroxy succinimide (NHS) is transferred to 250 ml flask fittedwith magnetic stirrer and nitrogen inlet. 200 ml dry benzene istransferred to the flask and about 50 ml of benzene is distilled off.The solution is cooled to 10° C. using an ice bath. 7.3 g triethylamineand 5.0 g fumaryl chloride are added dropwise to the NHS solution. Themixture is refluxed for 6 h under nitrogen atmosphere. At the end of a 6h period, the solution is cooled and filtered to remove triethylaminehydrochloride. The filtrate is concentrated by removing the solventunder vacuum and the crude product is recovered. The product is furtherpurified by column chromatography or recrystallization. The finalproduct is stored under nitrogen atmosphere at −20° C. until furtheruse.

32a-2) Synthesis of Fumaric Acid n-Hydroxysulfosuccinimide Ester(FUSNHS) Using 1,3-dicyclohexyl carbodiimide (DCC).

5 g fumaric acid is dissolved in 100 ml dry DMF. The solution is cooledto 4° C. 25.2 g 1,3-dicyclohexyl carbodiimide and 20.6 g ofN-hydroxysulfosuccinimide are added to the reaction mixture. The mixtureis stirred at 4° C. for 6 h and overnight at room temperature undernitrogen atmosphere. Dicyclohexylurea is removed by filtration and thefumaric acid NHS derivative is isolated by removing the DMF under vacuumand further purified by recrystallization or column chromatography. Theproduct is stored under nitrogen atmosphere at −20° C.

32b) Modification and Crosslinking of Tissue Using Fumaric Acidn-Hydroxysccinimide Ester (FUNHS).

Ten 1 cm by 1 cm bovine pericardium pieces, cut from a freshly obtainedbovine pericardial sac, are transferred to 50 ml polypropylenecentrifuge tube containing 10 ml phosphate buffered saline (PBS, (pH7.2). 300 mg of fumaric acid succinimide ester (FUNHS) dissolved in 0.5ml dimethyl sulfoxide is added to the tube and the solution is vortexedfor 15 minutes. The modification reaction is carried for 6 hours atambient temperature (25° C.) and then for 12 hours at 4° C. with gentleshaking. The reaction is terminated by washing the tissue with 20 ml PBS3 times. Finally, the tissue is stored in 30 ml 40% HEPES bufferedisopropanol solution. The tissue shows elevated shrink temperature ascompared to untreated tissue indicating crosslinking of the tissue,presumably due to crosslinking introduced by a reaction with abifunctional crosslinking agent. The crosslinks has polymerizableunsaturated group. The tissue is further crosslinked using free radicalpolymerizable monomer as described below:

32c) Additional Crosslinking of Fumaric Acid Modified Tissue UsingPhotopolymerization.

Two 1 cm by 1 cm pieces of fumaric acid crosslinked modified bovinepericardium tissue are incubated for 30 minutes each in 20% ethanol, 50%ethanol and 70% ethanol. 50 mg of 2,2-dimethoxy2-phenylacetophenone(Irgacure 651, free radical photoinitiator) is dissolved in 1 ml n-vinylpyrrolidinone to prepare a monomer solution. The 70% ethanol-incubatedtissue is transferred to photoinitiator solution and incubated for 4 hat 25° C. and then for 12 h at 0° C. The tissue is removed from solutionis placed on a glass plate and exposed to Black-Ray UV lamp (360 nmlight, 10000 mW/cm2 intensity) for 5 minutes from each side. Thecrosslinked tissue is removed and washed with 10 ml PBS to removeunreacted monomer, initiator fragments and water soluble non-crosslinkedpolymer.

Example 33 Implantable Tissue Surface Modification and/or CrosslinkingUsing Reagents Capable of Undergoing Hydrogen Abstraction Reaction orCapable of Initiating Free Radical Polymerization

a) Synthesis of Benzophenone Modified Polyethylene Glycol (BPEG)

A 3 necked 250 ml flask equipped with magnetic stirrer and nitrogeninlet is charged with 5 g 2-carboxyl benzophenone, 22 g polyethyleneglycol, molecular weight 1000 and 100 ml dichloromethane (DCM). Thesolution is cooled to 4° C. and 6.5 g 1,3-dicyclohexyl carbodiimide(DCC) are added under nitrogen atmosphere. The mixture is stirred at 4°C. for 6 h and overnight at room temperature under nitrogen atmosphere.Dicyclohexylurea is removed by filtration and thebenzophenone-terminated PEG ester is isolated by removing the DCM undervacuum. The PEG-benzophenone ester is further purified dissolving intoluene and precipitating in cold diethyl ether hexane.

b) Crosslinking/Surface Modification of Tissue Using by HydrogenAbstraction Mechanism

Two 1 cm by 1 cm pieces of bovine pericardium tissue samples areincubated for 30 minutes each in 20% ethanol, 50% ethanol and 70%ethanol and finally in 100% methanol. 200 mg benzophenone modifiedpolyethylene glycol (crosslinker) is dissolved in 1.8 ml methanol. Themethanol-incubated tissue is transferred to the crosslinker solution andincubated for 4 h at 25° C. and then for 12 h at 0° C. The tissue isremoved from solution and the placed on a quartz glass plate and exposedto high intensity UV lamp (360 nm) for 15 minutes on each side. Thecrosslinked/PEG surface modified tissue is removed and washed with 10 mlethanol to remove unreacted reagent.

Example 34 Chemical Modification of Tissue Using Benzophenone (HydrogenAbstraction Moiety) and then Crosslinking Using UV Light

a) Preparation of Protein Reactive Benzophenone Derivative (Bezophenonen-Hydroxysulfosuccinimide Ester)

A 3 necked 250 ml flask equipped with magnetic stirrer and nitrogeninlet is charged with 5 g 2-carboxyl benzophenone, 5.2 gn-hydroxysulfosuccinimide (SNHS) and 100 dry dimethylformamide (DMF) areadded. The solution is cooled to 4° C. and 6.5 g 1,3-dicyclohexylcarbodiimide (DCC) is added under nitrogen atmosphere. The mixture isstirred at 4° C. for 6 h and overnight at room temperature undernitrogen atmosphere. Dicyclohexylurea is removed by filtration andbezophenone SNHS ester is by isolated by removing the DMF under vacuum.The bezophenone SNHS ester is further purified by flash chromatography.

b) Tissue Modification Using Benzophenone SNHS Derivative.

Porcine pericardial sac is obtained from a local abbotair and is cleanedto remove blood and fatty tissue contaminants from the tissue surface.Ten 1 cm by 1 cm pieces are cut from the cleaned pericardial sac and aretransferred to 10 ml PBS solution (pH 7.2). 0.5 g bezophenone SNHS isadded to the buffered solution and the reaction is vortexed for 15minutes and continued for 6 hours at ambient temperature. An additional0.1 g benzophenone SNHS ester is added to the mixture at every 1.5hours. After the reaction, the tissue is isolated and washed severaltimes with PBS and ethanol solution finally with 100% methanol. Themodified tissue is then immediately used in tissue modificationreaction.

c) Crosslinking of Benzophenone Modified Tissue.

Two 1 cm by 1 cm pieces of benzophenone modified porcine pericardiumtissue samples are incubated for 30 minutes each in 20% ethanol, 50%ethanol, 70% ethanol, and finally in 100% methanol. The tissue isremoved from solution is placed on a quartz glass plate and exposed toUV lamp for 5 minutes from each side. The crosslinked/PEG surfacemodified tissue is removed and washed with 10 ml ethanol to removeunreacted reagent.

Example 35 Bioprosthetic Tissue Based Adhesive Patch for SurgicalApplications

Use of Membrane Like Implantable Tissue to Make a Surgical AdhesivePatch

Bovine pericardial sac is obtained from a local abbotair and is cleanedto remove blood and fatty tissue contaminants from the tissue surface. 2cm by 5 cm size pieces are cut from the cleaned pericardial sac.Separately a photopolymerizable adhesive solution is prepared frompolyethylene glycol 400 diacrylate (PEG400DA monomer). Photoinitiator(Eosin 0.02% and triethanol amine 0.5%) is dissolved in the PEG400DAmonomer liquid and this solution is applied on the pericardial tissuestrip. The monomer coated/incubated tissue serves as an adhesive patch.The adhesive coated patch is applied and conformed to the tissuegeometry on a surgical site such as, by way of example, and notlimitation, air-leaking lung tissue. The patch is applied in such a waythat the liquid adhesive layer is between the pericardial tissue andlung tissue. The liquid adhesive is cured or photopolymerized by argonion laser or xenon light emitting at 514 nm light. The green lightpasses through the pericardial tissue and initiates the polymerizationof PEGDA. The polymerized PEGDA adheres to the lung and pericardialtissue. If needed, the patch may also be immobilized by suturing on thelung tissue. The cured layer of PEGDA prevents air leak from the airtissue. If an absorbable patch is desired, then degradable implantabletissue (for example uncrosslinked bovine pericardial tissue) may, forexample, and without limitation, be used along with absorbable surgicaladhesive such as, by way of example, and not limitation, FocalSeal®marketed by Genzyme Biosurgery or DuraSeal™ surgical adhesive marketedby Confluent Surgcial Inc. Other surgical adhesives such as cynoacrylatebased adhesives may also be used.

Example 36 Radio-Opaque Implantable Tissue

Tissue Modification Using Iodinated Compound

a) Synthesis of N-Hydroxysuccinimide Ester of Triiodobenzoic Acid(TIBA-NHS)

1.2 g n-hydroxy succinimide and 1.1 g of triethyl amine are dissolved in100 ml benzene. The solution is cooled to 0° C. in ice bath. 1.2 gtriiodobenzoyl chloride is added dropwise to the cold alcohol solution.The mixture is then refluxed under nitrogen atmosphere for 3 h. Thesolution is filtered to remove triethylamine hydrochloride. The ester isthen isolated by removing the solvent. It is further purified by columnchromatography.

b) Chemical Bonding of Triiodobenzoic Acid Derivative to the Tissue.

Modification of Bovine Pericardium Tissue.

Ten 1 cm by 1 cm bovine pericardium pieces, cut from a freshly obtainedbovine pericardial sac, are transferred to 50 ml conical flaskcontaining 10 ml phosphate buffered saline (PBS, (pH 7.2). 250 mgtriiodobenzoic acid succinimide ester, TIBA-NHS ester dissolved in 0.5ml dimethyl sulfoxide is added to the tube and the solution is vortexedfor 15 minutes. 0.1 g of TIBA-NHS in 0.1 ml DMSO is added to thefixation solution every 2 hours up to six hours. The modificationreaction is carried for 6 hours at ambient temperature (25° C.) and thenfor 12 hours at 4° C. with gentle shaking. The reaction is terminated bywashing the tissue with 20 ml PBS 3 times. Finally, the tissue is storedin 30 ml 38% isopropanol and 2% benzyl alcohol solution at 4° C. untilfurther use. The triiodobenzoic acid moieties incorporated in the tissuemakes the tissue radio-opaque when viewed using medical x-ray imagingtechniques.

Alternatively radio-opaque compounds such as, by way of example, and notlimitation, iohexyl, metrizamide, iopamidol, iopentol, iopromide, andIoversol are added in the tissue coating formulations mentioned above.For example, Metrizamide 0.2 g and polylactic acid (0.8 g, molecularweight 50000) are dissolved in 10 ml chloroform and the polymer solutionis spray-coated or dip-coated on the tissue surface. The solvent isremoved by vacuum drying. The Metrizamide trapped inside the tissuecoating makes the tissue radio-opaque when viewed using medical x-rayimaging technique.

Radio-opaque tissue is also made by treating the tissue withcommercially available x-ray contrast agents such as, by way of example,and not limitation, Iohexyl or Ioversol in presence of EDC as a catalystand n-hydroxysuccinimide as a cocatalyst. The reaction is performed atpH 6.5 using PBS or other suitable buffers.

Tissue may also be crosslinked using a crosslinker containingradio-opaque groups such as, by way of example, and not limitation,triiodobenzene moieties.

Example 37 Crosslinking of Tissue Using Free Radical PolymerizableMonomers

Tissue Fixation Using Glycidyl Methacrylate and Subsequent Free RadicalPhotopolymerization Using Free Radical Polymerizable Monomers

Step 1

Treatment of Bovine or Sheep Pericardial Tissue with GlycidylMethacrylate.

Five pieces of unfixed tissue are attached to circular immobilization 28mm diameter ring clamps. The clamped tissues are suspended in 500 mlbeaker containing 200 ml PBS, 25 ml glycidyl methacrylate and 25 mltriethyl amine. The solution was stirred for 16 hours using magneticneedle stirrer. The beaker was transferred to a water bath maintained at50° C. and kept there for 4 hours. The tissue was then removed from theclamps washed with PBS and used in next step for treatment with freeradical polymerizable monomer and subsequent polymerization andcrosslinking.

Step-2

Fixation of Glycidyl Methacrylate Treated Tissue Using Free RadicalPolymerization.

Fixation Using Free Radical Photopolymerization.

One piece of glycidyl methacrylate treated tissue was incubated in 15 mlof 70% isopropyl alcohol solution for half an hour in a glass tube. Thetissue was then transferred to 2 ml n-vinyl pyrrolidinone solutioncontaining 40 mg 2-2 dimethoxy-2 phenyl acetophenone as a free radicallong ultraviolet light photoinitiator and incubated for 1 hour atambient temperature (25-30° C.). Care was taken to completely expose allsurfaces of the tissue to monomer solution during incubation. The tissuewas removed from the monomer solution and then placed on glass plate.The tissue was then exposed to long wavelength ultraviolet light(Black-Ray UV lamp, 360 nm flood light, 10000 mW/cm2 intensity) at adistance of 10 cm for 5 min from both sides. The fixed tissue was thenwashed with PBS and stored in alcohol solution until further use. Usinga similar procedure to that described above, tissue was treated withacrylic acid monomer instead of vinyl pyrrolidinone monomer.

Example 38 Modification of Tissue or Collagen by Cyclic LactonePolymerization

Preparation of Tissue-Hydroxyacid or Collagen-Polyhydroxyacid Copolymer

a) Modification of Tissue or Collagen Sponge by Cyclic Lactone(Dl-Lactide)

Three 2 cm×2 cm piece is cut from a cleaned uncrosslinked bovinepericardial sac. The tissue pieces are incubated for 30 minutes each in100% acetone. The dried tissue is then transferred into 100 ml flask.The tissue is further dried at 50° C. under vacuum for 10 hours andweighed. 10 ml tetrahydrofuran (THF), 3.0 g of dl-lactide and 100 mg ofstannous octoate are charged into the flask. The solution is refluxedfor 24 hours to promote cyclic polymerization of lactide by hydroxylgroups of tissue proteins (hydroxy proline). At the end of the reaction,tissue is removed, washed with methylene chloride to remove lactidemonomer and unbound polylactide polymer. The tissue is dried andweighed. The increase in weight is attributed to polylactide chemicallybound to the tissue matrix. A similar reaction can be carried out on thelyophilized commercially available collagen sponge from Knesey Nash orglutaraldehyde fixed pericardial tissue. Other cyclic lactone monomerssuch L-lactide, caprolactone, glycolide, trimethylene carbonate,dioxanone may also be polymerized or copolymerized in place of lactideusing a similar method mentioned above.

Example 39 Modification of Proteins Using Cyclic Lactones

A 100 ml round bottom flask is flame-dried under vacuum for 2 hours. 2 gof pharmaceutical grade gelatin is then transferred the flask. Thegelatin is further dried at 50° C. under vacuum for 10 hours andweighed. 10.0 gram of dl-lactide and 100 mg of stannous octoate arecharged into the flask. The flask is then heated in an oil bath at 120°C. for 24 hours. The flask is cooled and 20 ml chloroform is added tothe flask. The product is incubated in chloroform for 3 hours to removemonomer and unbound dl-lactide from the product. The solvent is removedand the gelatin-lactide copolymer is dried under vacuum and weighed.Other proteins or glycosaminoglycans such as, by way of example, and notlimitation, albumin, elastin, fibrinogen, collagen or hyaluronic acidmay also be used in place of gelatin. Other cyclic lactone monomers suchL-lactide, caprolactone, glycolide, trimethylene carbonate, dioxanonemay also be polymerized or copolymerized in place of lactide using asimilar method mentioned above.

Alternatively, gelatin may also be reacted with hydroxy acids such alactic acid in water at 100° C. to form a copolymer.

Example 40 Preparation of Tissue with Biostable and BiodegradableRegions with Controlled Geometry

Bovine pericardium (BP) tissue pieces (approximately 25 mm dia, 3pieces) are cut from a fresh pericardium and transferred to a 100 mlflask containing 10 ml PBS, 1.25 ml triethylamine (TEA) and 1.25 mlglycidyl methacrylate (GM). The solution was heated to 50° C. withconstant stirring using magnetic stir bar and held there for 8 hours.The tissue strips are removed from the solution and washed with PBS,distilled water, & incubated in 70% isopropanol solution in water for 30minutes. Finally, strips are then transferred to 10 ml vinylpyrrolidinone monomer solution containing 500 ppm 2,2-dimethoxy 2-phenylacetophenone (photoinitiator) and incubated at room temperature (30° C.)for one hour. One vinyl pyrrolidinone-infused sample piece wastransferred on a glass plate. An aluminum foil (8 mm diameter, 250micron thick) was placed at the center of the monomer-infused tissue.The tissue was then exposed to 360 nm light (Black-Ray UV lamp, 360 nmflood light, 10000 mW/cm2 intensity at a distance of 10 cm) for 5minutes to polymerize the vinyl pyrrolidinone monomer and crosslink thetissue. The same treatment was repeated using aluminum foil from theother side of the tissue. The exposed tissue was removed and washed withPBS and 70% isopropanol to remove unreacted monomer, uncrosslinkedpolymer and initiator fragments. The uncrosslinked regions where lightcould not penetrate due to aluminum foil and initiatephotopolymerization was visually noticeable and had different “feel” tothe human hand when compared to crosslinked or light exposed tissue.Using a similar method to that described above, another piece of thevinyl pyrrolidinone infused tissue was covered with aluminum foil (arrowshape, 8 mm long and 3 mm thick) and the exposed to polymerization andcrosslinking using 360 nm light for 5 minutes. The tissue under aluminumfoil can be considered as dark control tissue as discussed previouslywhere no polymerization and crosslinking occurred. The arrow-imprintedtissue was subjected to pepsin digestion for 48 hours. After 48 hours oftreatment with pepsin, the dark control tissue (in the shape of arrow)was completely digested by the enzyme while light exposed or crosslinkedtissue remained completely intact or stayed biostable. This observationwas confirmed in vivo by implanting the arrow-shaped imprinted tissue ina rat subcutaneous cavity for 60 days. After 60 days, the dark controlarea of the tissue was (unfixed tissue) substantially or completelydigested by natural protease enzymes present in the animal body and anarrow-like digested area was clearly visible to the naked eye onexplanted samples. It is understood that a variety of geometries andshapes may be created inside the tissue by using various masks to blockthe light similar to photolithography technique used in semiconductorprocessing art. Other techniques such as, by way of example, and notlimitation, electron beam irradiation or scanning of 360 nm laser lighton selected areas may also be used to create a pattern inside the tissuesimilar to principles used in electron beam lithography. In that case, amask may not be necessary to create a pattern (i.e., the beam itself cancreate a pattern).

Example 41 Membrane Like Thin and/or Large Tissue Obtained Using TissueEngineering Art for Bioprosthesis Applications

a) Engineering a Tissue from as Scaffold

5 cm×5 cm size porous collagen tissue engineering scaffold obtained fromKnesey Nash is soaked in 70% ethanol (overnight) to sterilize thescaffold, washed with sterile PBS (15 minutes, 3 times) and soaked inMinimum Essential Medium (MEM) supplemented with aminoacids, antibioticsand 30% fetal bovine serum (FBS) for 1.5 h. The scaffold is thentransferred to a sterile tissue culture dish. Human smooth muscle cellsor fibroblast cells passaged in MEM/10% FBS are resuspended in MEM/10%FBS and approximately 200000 cells per square inch are carefully placedon the scaffold. The cells are allowed to adhere for 30 minutes beforeadding 0.75 ml of the same medium to each sample. After 24 hours ofincubation in a 5% carbon dioxide atmosphere at 98% humidity and 37° C.,the samples are transferred to new wells and fresh medium and cells areadded to the scaffold. The addition of cells and media and incubation iscontinued until a desired tissue thickness is achieved (1 to 2000microns). For large size tissue, a large scaffold is used.

Porous tissue engineering scaffolds known in the tissue engineering artbased on polyesters such as polylactide or polyglycolide or theircopolymers. Natural scaffolds such as porcine sub-mucosa tissue may alsobe used in some cases to obtain a desired tissue engineered tissue.

The generated may be decellularized and used in bioprosthesismanufacturing. The tissue may be crosslinked or modified usingglutaraldehyde, EDC and other methods known in the art or methodsdescribed in this invention prior to using it in bioprosthesis use.

In one embodiment of the present invention the composition of matterproduced includes an uncrosslinked biological tissue, wherein the tissueis chemically modified with unsaturated polymerizable groups.

In another embodiment of the present invention the composition of matterproduced includes a biological tissue modified with unsaturated groups,wherein the unsaturated groups are used in chemical crosslinking of thetissue.

In yet another embodiment of the present invention, the composition ofmatter produced includes biological tissue modified with unsaturatedgroups, wherein a cross-linked biological tissue is produced by treatingthe tissue under effective cross-linking condition comprising a freeradical initiator or photoinitiator. In one embodiment, the crosslinkingis done in presence of a mono- or polyunsaturated compound capable ofcopolymerizing with the unsaturated groups in the tissue.

In an alternate embodiment of the present invention, the composition ofmatter produced includes a biological tissue modified with unsaturatedgroups, wherein unsaturated groups are copolymerized with free radicalpolymerizable comonomers. The comonomers may include, but are notlimited to, functional monomers with reactive functional groups such as,by way of example, and not limitation, epoxide or isocyanate; monomerswith charged groups; monomers that undergo crosslinking andbiodegradation; monomers that produce thermosensitive polymers; monomerswith long alkyl chains; monomers that produce crystalline orsemicrystalline polymers; monomers that produce functional polymers uponhydrolysis such as, by way of example, and not limitation, polyvinylalcohol; monomers with radio-opaque moieties; monomers that produceelastomers, monomers that have phosphorylcholine groups, and fluorinatedmonomers

In yet an alternate embodiment of the present invention, the compositionof matter produced includes a biological tissue modified and crosslinkedwith unsaturated groups, wherein modified unsaturated groups and/orcrosslinks with unsaturated groups are copolymerized with free radicalpolymerizable comonomers.

In another alternate embodiment of the present invention, thecomposition of matter produced includes a biological tissue modifiedwith unsaturated groups, wherein unsaturated groups are copolymerizedwith free radical polymerizable comonomers having biodegradable orhydrolizable groups. The biodegradable monomers may be hydrophilic orhydrophobic.

In one embodiment of the present invention, a cross-linked biologicaltissue is produced by treating the tissue under effective cross-linkingconditions with a biodegradable crosslinker. In some embodiments, thebiodegradable crosslinker may be a solute in a fluid including asolvent.

In yet another alternate embodiment of the present invention, thecomposition of matter produced includes a biological tissue modifiedwith unsaturated groups, wherein a cross-linked biological tissue isproduced by treating the unsaturated group-modified tissue undereffective cross-linking conditions with an organic di- or poly-mercaptocompounds. In one embodiment, the di- or poly-mercapto organic compoundmay be a solute in a fluid comprising a solvent.

In an alternate embodiment of the present invention, the composition ofmatter produced includes a membrane-like biological tissue and asurgical adhesive. The membrane-like tissue and surgical adhesive areformulated to form a “surgical adhesive patch”. Though not required, thesurgical adhesive patch may be biodegradable in some applications.

In another embodiment of the present invention, the composition ofmatter produced includes a radio-opaque implantable animal tissue.

In an embodiment of the present invention, the biological tissueproduced has shape memory properties

In yet another embodiment of the present invention, certain parts orregions of the biological tissue are made biostable while the remainingparts of the tissue are made biodegradable. The biostable andbiodegradable regions within the tissue can be of any geometry.

In another embodiment of the present invention, a non-crosslinkeddegradable biological tissue is produced by treating the tissue undereffective treatment conditions with a monofunctional reagent capable ofreacting with primary amine groups on the tissue. In one embodiment, themonofunctional reagent may be a polyether derivative or an activatedacid derivative such as, by way of example, and not limitation, ann-hydroxysuccinimide derivative, a cyclic lactone such as, by way ofexample, and not limitation, glycolide or lactide, an isocyanatederivative, or an anhydride derivative.

In one embodiment of the present invention, a biostable or biodegradabletissue produced by treating the tissue under effective treatmentconditions with a cyclic lactone to produce a tissue-polylactone graftcopolymer.

In another embodiment of the present invention a biological tissue isproduced in which the biological tissue/synthetic biodegradable polymercomposite is produced by treating the tissue with a fluid comprisingsynthetic biodegradable polymer.

In another embodiment of the present invention a biological tissue isproduced in which the biological tissue/synthetic biodegradable polymercomposite comprises a synthetic biodegradable polymer, which ischemically bonded to the biological tissue.

In one embodiment of the present invention a degradable biologicaltissue/synthetic biodegradable polymer composite produced by treating anon-crosslinked tissue under effective treatment conditions with asynthetic biodegradable polymer. In one embodiment, the syntheticbiodegradable polymer is polylactone or polyhydroxyacid derivative in afluid comprising a solvent. The synthetic biodegradable polymer can alsobe a crosslinked polymer.

In an alternate embodiment of the present invention the biologicaltissue is produced by treating the tissue with a fluid comprisingsynthetic biodegradable polymer and a bioactive compound.

In an alternate embodiment of the present invention, a method forcross-linking a tissue, which method may be utilized for tissue fixationis achieved by steps that include, without limitation, linking at leasta portion of the free radical polymerizable groups on the tissue with acovalent bond and crosslinking the free radical polymerizable groupsusing free radical mechanism or cyclic dimerization (e.g., treating thetissue under effective crosslinking conditions with a compound having afree radical polymerizable group). In another embodiment of the presentinvention, a method of modifying a protein or mixture of proteins in thesolid state is achieved by steps that include, without limitation,covalently bonding at least a portion of the protein functional groupswith a free radical polymerizable group in a solid state; and treatingthe protein under effective cross-linking conditions with a compoundhaving a free radical polymerizable group.

In an yet another alternate embodiment of the present invention, amethod for cross-linking a tissue, which method may be utilized fortissue fixation is achieved by steps that include, without limitation,covalently linking compounds containing at least one free radicalpolymerizable group on the tissue and crosslinking the free radicalpolymerizable group(s) using a di- or poly-mercapto organic compounds.In one embodiment, the method comprises covalently bonding at least aportion of the tissue functional groups with unsaturated groups andtreating the tissue under effective cross-linking conditions with a di-or polymercapto compound.

In an alternate embodiment of the present invention, a method forcross-linking a tissue, which method may be utilized for tissuefixation, is achieved by steps that include, without limitation,covalently bonding at least a portion of the tissue functional groupswith at least one free radical polymerizable group and treating thetissue under effective crosslinking conditions with a compound having afree radical group and a biodegradable link. In one embodiment, themethod comprises crosslinking the tissue with compounds containing atleast one free radical polymerizable group and further crosslinking thefree radical polymerizable group using free radical chemistry such as,by way of example, and not limitation, free radical dimerization andpolymerization, free radical crosslinking or free radicalcopolymerization with monomer.

In an alternate embodiment of the present invention, the method formaking a biodegradable biological tissue includes the steps of withoutlimitation, treating the tissue under effective cross-linking conditionswith a fluid comprising a biodegradable crosslinker.

In one embodiment of the present invention, the method for incorporatinga biodegradable polymer in a biological tissue is achieved by steps thatinclude, without limitation, dehydrating the biological tissue; treatingthe dehydrated tissue with a solution of biodegradable polymer in anorganic solvent; and removing the solvent from the treated tissue.

In yet another embodiment of the present invention, the method forincorporating a biodegradable polymer and a bioactive compound in animplantable biological tissue is achieved by steps that include, withoutlimitation, dehydrating the biological tissue; treating dehydratedtissue with a solution of biodegradable polymer and bioactive compoundin an organic solvent; and removing the solvent from the treated tissue.In an alternate embodiment, the method comprises the steps of providinga tissue suitable for human implantation; exposing the tissue to a fluidcomprising sparingly soluble drug dissolved in a solvent; andevaporating the solvent.

In an alternate embodiment of the present invention, the biologicaltissue based controlled drug delivery patch that releases at least onebioactive compound.

In an alternate embodiment of the present invention, the method formaking an implantable degradable drug delivery patch from themembrane-like tissue is achieved by steps that include, withoutlimitation, dehydrating the membrane-like biological tissue; treatingdehydrated membrane-like tissue with a solution of biodegradable polymerand a bioactive compound in an organic solvent; removing the solventfrom the treated tissue; releasing the compound from the biodegradablepolymer. In an alternate embodiment, the method comprises providing aimplantable membrane-like animal tissue; exposing the tissue to a fluidcomprising controlled release carrier and a bioactive compound dispersedin the solvent; and evaporating the solvent. The bioactive compound maybe a cell cycle inhibitor including, but not limited to, Lovastatin(HMG-CoA inhibitor or statin), paclitaxel, and Rapamycin. Thebiodegradable polymer may be hydrophobic or hydrophilic. Thebiodegradable polymer can be a crosslinked polymer, though not required.

In an alternate embodiment of the present invention, the method forcoating the biological tissue with biodegradable polymer (e.g., forminga coated tissue or tissue implant) is achieved by steps that include,without limitation, providing a tissue suitable for human implantation;coating the tissue surface by spraying a solution comprising a solventand a polymer dissolved in the solvent; and evaporating the solvent. Inan alternate embodiment, the method further comprises the steps ofproviding a tissue suitable for human implantation; exposing the tissueto a fluid comprising biodegradable polymer dispersed in the solvent;and evaporating the solvent. In one embodiment, the method comprises thesteps of providing a tissue suitable for human implantation; coating thetissue surface by spraying a solution comprising precursors capable offorming crosslinked polymers; and crosslinking the precursors. Inanother alternate embodiment, the method comprises the steps ofproviding a tissue suitable for human implantation; coating the tissuesurface by spraying a macromonomer solution; and crosslinking themacromonomer in the solution. In one exemplary embodiment, the methodcomprises dehydrating the biological tissue; spraying a coating solutioncomprising biodegradable polymer in a solvent; and removing the solventfrom the treated tissue. In an alternate embodiment, the methodcomprises dehydrating the biological tissue; dipping the dehydratedtissue in a coating solution comprising biodegradable polymer in asolvent; removing the solvent from the treated tissue.

In another embodiment of the present invention, a method for making aradio-opaque implantable tissue is achieved by steps that include,without limitation, treating a biological tissue with a radio-opaquecompound under effective treatment conditions to covalently bond theradio-opaque compound to the tissue. In some embodiments, theradio-opaque compound is an iodinated organic compound.

In another embodiment of the present invention, a method for making aradio-opaque implantable tissue is achieved by steps that include,without limitation, treating a biological tissue with a radio-opaquecompound and biodegradable polymer in a solvent and removing the solventto produce a tissue coated with biodegradable compound and radio-opaquecompound.

Another embodiment of the present invention provides for a method oftreating a tissue under effective cross-linking conditions with a di- orpoly-mercapto organic compound.

In one embodiment of the present invention, a method of coating abiological implantable tissue with a biodegradable hydrogel is achievedby steps that include, without limitation, treating a tissue with aprecursor or biodegradable hydrogel components and crosslinking theprecursors to produce a biodegradable hydrogel coating on the surface ofthe tissue.

In another embodiment of the present invention, a method of coating abiological implantable tissue with biodegradable hydrogel comprisingcells/bioactive compound is achieved by steps that include, withoutlimitation, treating a tissue with a precursor or biodegradable hydrogelcomponents comprising cells and/or bioactive compounds and crosslinkingthe precursors to produce a biodegradable hydrogel coating withentrapped cells/drug in the coating on the surface of the tissue.

In yet another embodiment of the present invention, a method of coatinga biological tissue with non-crosslinked biodegradable hydrogel isachieved by steps that include, without limitation, dehydrating thebiological tissue; treating the dehydrated tissue with a solution ofbiodegradable polymer in an organic solvent; removing the solvent fromthe treated tissue; and exposing the tissue to a biological environmentto hydrate the tissue and biodegradable polymer.

In another embodiment of the present invention, a method forincorporating a biodegradable polymer and bioactive substance in abiological tissue is achieved by steps that include, without limitation,forming grooves or holes on tissue surface; filling the grooves or holeswith a biodegradable polymer and bioactive compound; and releasing thebioactive compound in a controlled manner.

In an alternate embodiment of the present invention a degradable animaltissue is coated with or incorporated with, Demineralized Bone Matrix(DBM) and/or purified bone morphogenic proteins (BMP). This mixtureprovides a matrix that allows the cellular components of the body tomigrate into it and thus produce osteoinduction where needed. The matrixcomposition, enzymes (such as, by way of example, and not limitation,thrombin and plasmin), BMPs, growth factors and DBM and theirconcentrations, calcium salts such as, by way of example, and notlimitation, calcium phosphates may be adequately formulated to optimizethe longevity of this temporal scaffolding structure and theosteoinduction which needs to occur. All of the animal tissue componentsare biodegradable, but during osteogenesis the mixture provides anon-collapsible scaffold that can determine the shape and location ofthe newly formed bone.

In one embodiment of the present invention the composition of matterproduced comprises a degradable tissue coated with a biodegradablepolymer comprising at least one growth factor and/or a drug.

In another embodiment of the present invention, the composition ofmatter including a biodegradable implantable animal tissue coated withbiodegradable polymer and an effective concentration of at least onegrowth factor, wherein the concentration of growth factor is effectivein promoting wound healing.

An alternate embodiment of the present invention provides for acomposition of matter that promotes the growth of cells, including adegradable animal tissue; a hydrogel coating on the surface ofdegradable tissue; and an effective concentration of at least one growthfactor, wherein the concentration of the growth factor is effective inpromoting the directed migration of the animal cells. In anotherembodiment, genetically altered cells and/or other cells may also beincluded in the tissue-coated hydrogels of this invention.

In one embodiment of the present invention, a composition of matter thatpromotes the proliferation and/or differentiation of animal cells ismade by including an implantable animal tissue; a hydrogel; and aneffective concentration of at least one growth factor, wherein theconcentration is effective in promoting proliferation and/ordifferentiation of animal cells.

In an alternate embodiment of the present invention, the composition ofmatter produced is used to promote the localized delivery of at leastone growth factor. Exemplary, but not limiting, growth factors arevascular endothelial growth factor (VEGF) or BMP or mixtures thereof.The use of numerous other growth factors, both known and yet to bediscovered, will be readily apparent to one skilled in the art, in lightof the teachings of the present invention.

One embodiment of the present invention provides for a process forpromoting the healing of wounds, which is achieved by steps thatinclude, without limitation, applying to the wound a composition thatcontains a non-crosslinked animal degradable animal tissue modified witha synthetic polymer and an effective concentration of at least onegrowth factor or one small molecule therapeutic, wherein theconcentration is effective to promote wound healing.

In one embodiment of the present invention, a degradable implantableanimal tissue-based composition produced is used to promote thelocalized delivery of a poorly water-soluble form of a bioactivecompound, such as, but not limited to, chlorhexidene; chlorhexidenediacetate monohydrate or chlorhexidene dihydrochloride; chlorhexidenegluconate, silver salts such as, by way of example, and not limitation,silver chloride, silver iodide, silver acetate, silver lactate, cellcycle inhibitors such as, by way of example, and not limitation,paclitaxel, lovastatin, rapamycin, simvastatin, rifampin; oranti-arrhythmic agents such as, by way of example, and not limitation,amiodarone.

In another embodiment of the present invention, the method for tissuecrosslinking or fixation is achieved by steps that include, withoutlimitation, linking the free radical polymerizable groups on the tissuewith a covalent bond and crosslinking the free radical polymerizablegroups using free radical polymerizable monomers including a primaryamine group. Further crosslinking the primary amine groups using a di-or polyfunctional crosslinker such as, by way of example, and notlimitation, glutaraldehyde.

In one embodiment of this of invention a bioprosthesis made usingmembrane like tissue is provided wherein the membrane like tissue ismade using tissue engineering methodologies and has a thickness from 10micron to 2000 microns and/or has a size >1 square inch.

In an alternate embodiment of the present invention, the method formaking a degradable tissue matrix is achieved by steps that include,without limitation, substantially water-insoluble drug or bioactivecompound is provided for in an alternate embodiment of the presentinvention. The method includes dehydrating the membrane-like biologicaltissue; treating dehydrated membrane-like tissue with a solution of asubstantially water-insoluble bioactive compound in an organic solvent;and removing the solvent from the treated tissue.

Having fully described at least one embodiment of the present invention,other equivalent or alternative implantable tissue composition andmethods will be apparent to those skilled in the art. The invention hasbeen described above by way of illustration, and the specificembodiments disclosed are not intended to limit the invention to theparticular forms disclosed. The invention is thus to cover allmodifications, equivalents, and alternatives falling with the spirit andscope of the following claims.

1. A biodegradable bioprosthesis comprising: a first biodegradabletissue portion; a second biodegradable tissue portion; and one or morebiodegradable crosslinkers crosslinking the first biodegradable tissueportion with the second biodegradable tissue portion.
 2. Thebioprosthesis of claim 1, wherein the first biodegradable tissue portionand/or the second biodegradable tissue portion includes animal tissue.3. The bioprosthesis of claim 1, wherein the one or more biodegradablecrosslinkers degrade by hydrolysis or enzyme.
 4. The bioprosthesis ofclaim 3, wherein the first biodegradable tissue portion and/or thesecond biodegradable tissue portion becomes susceptible to enzymaticdegradation upon degradation of the one or more biodegradablecorsslinkers.
 5. The bioprosthesis of claim 4, wherein the wherein thefirst biodegradable tissue portion and/or the second biodegradabletissue portion degrades into non-toxic components.
 6. The bioprosthesisof claim 1, wherein the one or more biodegradable crosslinkers have twoor more functional groups reacted with the first biodegradable tissueportion and/or the second biodegradable tissue portion and one or morebiodegradable segments having a biodegradable covalent bond.
 7. Thebioprosthesis of claim 6, wherein the two or more functional groupsinclude a reaction product of an anhydride, isocyanate, epoxy,n-hydroxysuccinimide, or aldehyde reacted with a tissue.
 8. Thebioprosthesis of claim 7, wherein the two or more functional groups arecovalently coupled to collagen or elastin of the first biodegradabletissue portion and/or the second biodegradable tissue portion.
 9. Thebioprosthesis of claim 6, wherein the one or more biodegradable segmentsinclude an ester or amide bond that undergoes cleavage underphysiological conditions.
 10. The bioprosthesis of claim 6, wherein theone or more biodegradable segments include a cleavable polymer,copolymer, or oligomer of glycolide, dl-lactide, l-lactide,caprolactone, dioxanone, or trimethylene carbonate.
 11. Thebioprosthesis of claim 6, wherein the one or more biodegradable segmentsinclude a peptidic linkage cleavable by metalloproteinases and/orcollagenease enzymes.
 12. The bioprosthesis of claim 6, wherein the oneor more biodegradable segments include poly(hydroxy acid),poly(orthocarbonate), poly(anhydride), poly(lactone), poly(aminoacid),poly(carbonate), poly(phosphonate), and copolymers thereof.
 13. Thebioprosthesis of claim 6, wherein the one or more biodegradable segmentsinclude a succinate, glutarate, or itaconate.
 14. The bioprosthesis ofclaim 6, wherein the one or more biodegradable segments includepolyethylene glycol.
 15. The bioprosthesis of claim 6, wherein the firstbiodegradable tissue portion and/or the second biodegradable tissueportion are from the same tissue.
 16. The bioprosthesis of claim 6,wherein the first biodegradable tissue portion and/or the secondbiodegradable tissue portion are from one or more tissues selected fromthe group consisting of, decellularized tissue, chemically modifiedtissue, cultured tissue from mammalian cells and crosslinked tissue. 17.A method of making the biodegradable bioprosthesis of claim 1, themethod comprising: providing the first biodegradable tissue portion andthe second biodegradable tissue portion; providing the one or morebiodegradable crosslinkers; and crosslinking the first biodegradabletissue portion and the second biodegradable tissue portion with the oneor more biodegradable crosslinkers.
 18. The method of claim 17, whereinthe crosslinking is in accordance with one or more of the following: ata temperature from about 0° C. to about 55° C.; at a pH from about 6 toabout 9; crosslinked without denaturing the tissue; or in a bufferedaqueous solution.
 19. A medical device comprising the biodegradablebioprosthesis of claim
 1. 20. A controlled drug delivery systemcomprising the biodegradable bioprosthesis of claim 1.